GNAQ targeted dsRNA compositions and methods for inhibiting expression

ABSTRACT

The invention relates to a double-stranded ribonucleic acid (dsRNA) targeting a G-alpha q subunit (GNAQ) of a heterotrimeric G gene, and methods of using the dsRNA to inhibit expression of GNAQ.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/507,086, filed Oct. 6, 2014, allowed, which is a continuation of U.S.patent application Ser. No. 13/614,019, filed Sep. 13, 2012, now U.S.Pat. No. 8,889,644, issued Nov. 18, 2014, which is a continuation ofU.S. patent application Ser. No. 12/635,630, filed Dec. 10, 2009, nowU.S. Pat. No. 8,324,368, issued Dec. 4, 2012, which claims the benefitof U.S. Provisional Application No. 61/121,253, filed Dec. 10, 2008, andU.S. Provisional Application No. 61/185,543, filed Jun. 9, 2009, andU.S. Provisional Application No. 61/244,780, filed Sep. 22, 2009, whichare hereby incorporated in their entirety by reference.

REFERENCE TO A SEQUENCE LISTING

This application includes a Sequence Listing which contains 1762sequences and submitted electronically as a text file named35643_US_sequencelisting.txt, created on Dec. 5, 2016, with a size of560,474 bytes. The sequence listing is incorporated by reference.

FIELD OF THE INVENTION

The invention relates to a double-stranded ribonucleic acid (dsRNA)targeting a G-alpha q subunit (GNAQ) of a heterotrimeric G gene, andmethods of using the dsRNA to inhibit expression of GNAQ.

BACKGROUND OF THE INVENTION

Guanine nucleotide-binding proteins (G proteins) are a family ofheterotrimeric proteins that couple cell surface, 7-transmembrane domainreceptors to intracellular signaling pathways. G proteins are composedof alpha, beta and gamma subunits. The G-alpha q subunit (GNAQ) is oneof the G-alpha subunits. GNAQ mediates stimulation of phospholipaseC-beta and hydrolysis of GTP.

Mice with GNAQ mutations leading to overexpression of GNAQ exhibitdermal hyperpigmentation. A point mutation in human GNAQ was reported ina melanoma sample (Bamford et al (2004) Br J Cancer, 91:355-358). InWO/2008/098208 (PCT/US2008/053484), the Applicant's described thepresence of mutations that constitutively activate GNAQ in melanocyticneoplasms, e.g., uveal melanomas.

Double-stranded RNA molecules (dsRNA) have been shown to block geneexpression in a highly conserved regulatory mechanism known as RNAinterference (RNAi). WO 99/32619 (Fire et al.) disclosed the use of adsRNA of at least 25 nucleotides in length to inhibit the expression ofgenes in C. elegans. dsRNA has also been shown to degrade target RNA inother organisms, including plants (see, e.g., WO 99/53050, Waterhouse etal.; and WO 99/61631, Heifetz et al.), Drosophila (see, e.g., Yang, D.,et al., Curr. Biol. (2000) 10:1191-1200), and mammals (see WO 00/44895,Limmer; and DE 101 00 586.5, Kreutzer et al.).

SUMMARY OF THE INVENTION

Disclosed herein are dsRNAs targeted to GNAQ for inhibiting expressionof GNAQ in a cell. Also disclosed are methods of using the GNAQ dsRNAfor siRNA inhibition of GNAQ expression and treatment of diseaseassociated with expression and/or over expression of GNAQ, e.g., uvealmelanoma.

Accordingly one aspect of the invention is a double-stranded ribonucleicacid (dsRNA) for inhibiting expression of a G-alpha q subunit (GNAQ) ofa heterotrimeric G gene, having a sense strand and an antisense strandhaving a region of complementarity complementary to an mRNA encodingGNAQ, wherein each strand is at least 15 nucleotides in length. In oneembodiment the dsRNA is AD-20057, e.g., sense strand is SEQ ID NO:1579and the antisense strand is SEQ ID NO:1580. In another embodiment, theantisense strand is complementary to at least 15 contiguous nucleotidesof SEQ ID NO:1421 or is complementary to at least the first 11nucleotides of SEQ ID NO:1421. The sense strand can include 15 or morecontiguous nucleotides of SEQ ID NO:1421 or SEQ ID NO:1579 and/or theantisense strand can include 15 or more contiguous nucleotides of SEQ IDNO:1422 or SEQ ID NO:1580. In some embodiments the sense strandnucleotide sequence includes SEQ ID NO:1421 and the antisense strandnucleotide sequence includes SEQ ID NO:1422.

In some embodiments the dsRNA of the invention results in the following:administration of 0.1 nM of the dsRNA to a A375 cell results in about66% inhibition of GNAQ mRNA expression as measured by a real time PCRassay or administration of 1 nM of the dsRNA to a A375 cell results inabout 61% inhibition of GNAQ mRNA expression as measured by a real timePCR assay or administration of 1 nM of the dsRNA to a A579 cell resultsin about 82% inhibition of GNAQ mRNA expression as measured by a realtime PCR assay or administration of 10 nM of the dsRNA to a OMM1.3 cellresults in about 42% inhibition of GNAQ mRNA expression as measured by areal time PCR assay or administration of the dsRNA to a UMEL202 cellresults in about 81% inhibition of GNAQ mRNA expression as measured by areal time PCR assay.

In another embodiment, the dsRNA is AD-20051 and the sense strand is SEQID NO:1565 and the antisense strand is SEQ ID NO:1566. The dsRNA can becomplementary to at least the first 11 nucleotides of SEQ ID NO:1407and/or complementary to at least 15 contiguous nucleotides of SEQ IDNO:1407. In some embodiments the sense strand includes 15 or morecontiguous nucleotides of SEQ ID NO: 1407 or SEQ ID NO:1565 and/or theantisense strand includes 15 or more contiguous nucleotides of SEQ IDNO:1408 or SEQ ID NO:1566. The sense strand nucleotide sequence caninclude SEQ ID NO:1407 and the antisense strand nucleotide sequence caninclude SEQ ID NO:1408.

In some embodiments the dsRNA of the invention results in the following:administration of 0.1 nM of the dsRNA to a A375 cell results in about49% inhibition of GNAQ mRNA expression as measured by a real time PCRassay or administration of 1 nM of the dsRNA to a A375 cell results inabout 55% inhibition of GNAQ mRNA expression as measured by a real timePCR assay or administration of 1 nM of the dsRNA to a A579 cell resultsin about 83% inhibition of GNAQ mRNA expression as measured by a realtime PCR assay or administration of 10 nM of the dsRNA to a OMM1.3 cellresults in about 42% inhibition of GNAQ mRNA expression as measured by areal time PCR assay.

In other embodiments the dsRNA is AD-20052 or AD-20069.

The antisense strand of the dsRNA is partially or completelycomplementary to an mRNA encoding a GNAQ, e.g., to a human GNAQ mRNA(e.g., NM 002072) or to a rat GNAQ mRNA (e.g., NM_031036). The regioncomplementary is at least 15 nucleotides in length, e.g., between 19 and21 nucleotides in length, e.g., 19 nucleotides in length. The region ofcomplementarity can include at least 15 contiguous nucleotides of one ofthe antisense sequences listed in Tables 2a, 3a, or 4a. In otherembodiments, the region of complementarity is one of the antisensesequences listed in Tables 2a, 3a, or 4a.

Additional exemplary dsRNA are provided in the tables herein. In someembodiments, the dsRNA of the invention includes a sense strand andantisense strand are selected from Tables 2b, 3b, 4b or Tables 2c, 3c,or 4c or Tables 2d, 3d, or 4d.

In one aspect, each strand of the dsRNA is no more than 30 nucleotidesin length. At least one strand can include a 3′ overhang of at least 1nucleotide, e.g., 2 nucleotides, e.g., dTdT.

In some embodiments, the dsRNA is modified. For example, the dsRNA caninclude a modification that causes the dsRNA to have increased stabilityin a biological sample. In one embodiment, the dsRNA includes at leastone modified nucleotide, e.g., a 2′-O-methyl modified nucleotide, anucleotide comprising a 5′-phosphorothioate group, or a terminalnucleotide linked to a cholesteryl derivative or dodecanoic acidbisdecylamide group. In other embodiments the modified nucleotide is a2′-deoxy-2′-fluoro modified nucleotide, a 2′-deoxy-modified nucleotide,a locked nucleotide, an abasic nucleotide, 2′-amino-modified nucleotide,2′-alkyl-modified nucleotide, morpholino nucleotide, a phosphoramidate,or a non-natural base comprising nucleotide. The dsRNA of the inventioncan include at least one 2′-O-methyl modified nucleotide and at leastone 2′-deoxythymidine-3′-phosphate nucleotide comprising a5′-phosphorothioate group.

Any of the dsRNA of the invention can be modified according to a set ofrules, e.g., the sense strand includes all 2′-O-methyl modifiedpyrimidines and the antisense strand comprises 2′-O-methyl modifiedpyrimidines when the pyrimidine is adjacent to A and each strandcomprises dTdT at the 3′ end or the sense strand comprises all2′-O-methyl modified pyrimidines and the antisense strand comprises2′-O-methyl modified pyrimidines when the pyrimidine is adjacent to Aand each strand comprises dTsdT at the 3′ end or the sense strandcomprises all 2′-O-methyl modified pyrimidines and the antisense strandcomprises 2′-O-methyl modified pyrimidines when a) the pyrimidine isadjacent to A or b) the pyrimidine is a uracil adjacent to a U or a G,and each strand comprises dTsdT at the 3′ end.

In some embodiments the dsRNA include a ligand. The ligand can beconjugated to the 3′-end of the sense strand of the dsRNA.

Another aspect of the invention is a composition for inhibitingexpression of a GNAQ gene including a dsRNA targeting GNAQ and apharmaceutical formulation. In one embodiment, the pharmaceuticalformulation is a lipid formulation. Exemplary formulations are describedherein and include, for example, a LNP formulation, a LNP01 formulation,a XTC-SNALP formulation, a SNALP formulation, or a LNP11 formulation.

Also included herein is an isolated cell containing a dsRNA of theinvention, a vector including the nucleotide sequence that encodes atleast one strand of the dsRNA of the invention, and a cell includingsaid vector.

A dsRNA of the invention, upon contact with a cell expressing said GNAQ,inhibits expression of said GNAQ gene by at least 40% compared to a cellnot so contacted. In some embodiments, a dsRNA of the invention has a pMIC50, e.g., an IC50 of less than 10 pM.

Another aspect of the invention is method of inhibiting GNAQ expressionin a cell, the method including introducing into the cell any of thedsRNA of the invention and maintaining the cell for a time sufficient toobtain degradation of the mRNA transcript of a GNAQ gene, therebyinhibiting expression of the GNAQ gene in the cell. In some embodiments,expression is inhibited by at least 20%, 40%, 60%, or at least 80%. Alsoincluded is a method of treating a disorder mediated by GNAQ expressionby administering to a human in need of such treatment a therapeuticallyeffective amount of any of the dsRNA of the invention. Examples of saiddisorders include uveal melanoma, cutaneous melanoma, Blue nevi, Nevi ofOta, a small lung tumor, or a neuroendocrine tumors. The method oftreatment can include administering an addition composition, e.g., asecond dsRNA.

DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing IFN-alpha cytokine induction in human PBMCsfollowing transfection with a set of GNAQ targeted dsRNA.

FIG. 2 shows TNF-alpha cytokine induction in human PBMCs followingtransfection with a set of GNAQ targeted dsRNA.

FIG. 3 shows cell viability of OMM1.3 and MEL285 cells followingtransfection with 1 nM of dsRNAs. The Y-axis is viability normalized tocontrol AD-1955.

FIG. 4 shows cell viability of MEL202 and MEL285 cells followingtransfection with 1 nM of dsRNAs. The Y-axis is viability normalized tocontrol AD-1955.

FIG. 5 shows cell viability of OMM1.3 and MEL285 cells followingtransfection with 0.01 nM of dsRNAs. The Y-axis is viability normalizedto control AD-1955.

FIG. 6 shows cell viability of MEL202 and MEL285 cells followingtransfection with 0.01 nM of dsRNAs. The Y-axis is viability normalizedto control AD-1955.

FIG. 7 shows day 7 cell viability of OMM1.3, MEL202, and MEL285 cellsfollowing transfection with AD-20057 and AD-20051 dsRNAs

FIG. 8 shows day 7 cell viability of OMM1.3, MEL202, and MEL285 cellsfollowing transfection with AD-20069 and AD-20093 dsRNAs.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides dsRNAs and methods of using the dsRNAs forinhibiting the expression of a G-alpha q subunit (GNAQ) of aheterotrimeric G gene in a cell or a mammal where the dsRNA targets aGNAQ gene. The invention also provides compositions and methods fortreating pathological conditions and diseases, such as uveal melanoma ina mammal caused by the over-expression of a GNAQ gene. A dsRNA directsthe sequence-specific degradation of mRNA through a process known as RNAinterference (RNAi).

The dsRNAs of the compositions featured herein include an antisensestrand having a region which is less than 30 nucleotides in length,generally 19-24 nucleotides in length, and is complementary to at leastpart of an mRNA transcript of a GNAQ gene. The use of these dsRNAsenables the targeted degradation of mRNAs of genes that are implicatedin pathologies associated with GNAQ expression in mammals. Very lowdosages of GNAQ dsRNAs in particular can specifically and efficientlymediate RNAi, resulting in significant inhibition of expression of aGNAQ gene. Using cell-based assays, the present inventors demonstratethat dsRNAs targeting GNAQ can specifically and efficiently mediateRNAi, resulting in significant inhibition of expression of a GNAQ gene.Thus, methods and compositions including these dsRNAs are useful fortreating pathological processes that can be mediated by down regulatingGNAQ over-expression, such as, e.g., treatment of uveal melanoma.

The following detailed description discloses how to make and use thecompositions containing dsRNAs to inhibit the expression of a GNAQ gene,as well as compositions (e.g., pharmaceutical compositions) and methodsfor treating diseases and disorders caused by the expression of thisgene.

Accordingly, in some aspects, pharmaceutical compositions containing aGNAQ dsRNA and a pharmaceutically acceptable carrier, methods of usingthe compositions to inhibit expression of a GNAQ gene, and methods ofusing the pharmaceutical compositions to treat diseases caused byexpression of a GNAQ gene are featured in the invention.

Definitions

For convenience, the meaning of certain terms and phrases used in thespecification, examples, and appended claims, are provided below. Ifthere is an apparent discrepancy between the usage of a term in otherparts of this specification and its definition provided in this section,the definition in this section shall prevail.

“G,” “C,” “A” and “U” each generally stand for a nucleotide thatcontains guanine, cytosine, adenine, and uracil as a base, respectively.“T” and “dT” are used interchangeably herein and refer to adeoxyribonucleotide wherein the nucleobase is thymine, e.g.,deoxyribothymine. However, it will be understood that the term“ribonucleotide” or “nucleotide” or “deoxyribonucleotide” can also referto a modified nucleotide, as further detailed below, or a surrogatereplacement moiety. The skilled person is well aware that guanine,cytosine, adenine, and uracil may be replaced by other moieties withoutsubstantially altering the base pairing properties of an oligonucleotidecomprising a nucleotide bearing such replacement moiety. For example,without limitation, a nucleotide comprising inosine as its base may basepair with nucleotides containing adenine, cytosine, or uracil. Hence,nucleotides containing uracil, guanine, or adenine may be replaced inthe nucleotide sequences of the invention by a nucleotide containing,for example, inosine. Sequences comprising such replacement moieties areembodiments of the invention.

As used herein, “GNAQ” refers to a G-alpha q subunit (GNAQ) of aheterotrimeric G gene. GNAQ is also known as guanine nucleotide bindingprotein (G protein), q polypeptide and G-ALPHA-q, GAQ. The sequence of ahuman GNAQ mRNA transcript can be found at NM_002072.2. The sequence ofrat GNAQ mRNA can be found at NM_031036.

A used herein “target” or “target gene” refers to a GNAQ gene.

As used herein, “target sequence” refers to a contiguous portion of thenucleotide sequence of an mRNA molecule formed during the transcriptionof a GNAQ gene, including mRNA that is a product of RNA processing of aprimary transcription product.

As used herein, the term “strand comprising a sequence” refers to anoligonucleotide comprising a chain of nucleotides that is described bythe sequence referred to using the standard nucleotide nomenclature.

As used herein, and unless otherwise indicated, the term“complementary,” when used to describe a first nucleotide sequence inrelation to a second nucleotide sequence, refers to the ability of anoligonucleotide or polynucleotide comprising the first nucleotidesequence to hybridize and form a duplex structure under certainconditions with an oligonucleotide or polynucleotide comprising thesecond nucleotide sequence, as will be understood by the skilled person.Such conditions can, for example, be stringent conditions, wherestringent conditions may include: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mMEDTA, 50° C. or 70° C. for 12-16 hours followed by washing. Otherconditions, such as physiologically relevant conditions as may beencountered inside an organism, can apply. The skilled person will beable to determine the set of conditions most appropriate for a test ofcomplementarity of two sequences in accordance with the ultimateapplication of the hybridized nucleotides.

This includes base-pairing of the oligonucleotide or polynucleotidecomprising the first nucleotide sequence to the oligonucleotide orpolynucleotide comprising the second nucleotide sequence over the entirelength of the first and second nucleotide sequence. Such sequences canbe referred to as “fully complementary” with respect to each otherherein. However, where a first sequence is referred to as “substantiallycomplementary” with respect to a second sequence herein, the twosequences can be fully complementary, or they may form one or more, butgenerally not more than 4, 3 or 2 mismatched base pairs uponhybridization, while retaining the ability to hybridize under theconditions most relevant to their ultimate application. However, wheretwo oligonucleotides are designed to form, upon hybridization, one ormore single stranded overhangs, such overhangs shall not be regarded asmismatches with regard to the determination of complementarity. Forexample, a dsRNA comprising one oligonucleotide 21 nucleotides in lengthand another oligonucleotide 23 nucleotides in length, wherein the longeroligonucleotide comprises a sequence of 21 nucleotides that is fullycomplementary to the shorter oligonucleotide, may yet be referred to as“fully complementary” for the purposes described herein.

“Complementary” sequences, as used herein, may also include, or beformed entirely from, non-Watson-Crick base pairs and/or base pairsformed from non-natural and modified nucleotides, in as far as the aboverequirements with respect to their ability to hybridize are fulfilled.Such non-Watson-Crick base pairs includes, but not limited to, G:UWobble or Hoogstein base pairing.

The terms “complementary,” “fully complementary” and “substantiallycomplementary” herein may be used with respect to the base matchingbetween the sense strand and the antisense strand of a dsRNA, or betweenthe antisense strand of a dsRNA and a target sequence, as will beunderstood from the context of their use.

As used herein, a polynucleotide that is “substantially complementary toat least part of” a messenger RNA (mRNA) refers to a polynucleotide thatis substantially complementary to a contiguous portion of the mRNA ofinterest (e.g., a target gene, e.g., an mRNA encoding GNAQ) including a5′ UTR, an open reading frame (ORF), or a 3′ UTR. For example, apolynucleotide is complementary to at least a part of a GNAQ mRNA if thesequence is substantially complementary to a non-interrupted portion ofan mRNA encoding GNAQ.

The term “double-stranded RNA” or “dsRNA,” as used herein, refers to acomplex of ribonucleic acid molecules, having a duplex structurecomprising two anti-parallel and substantially complementary, as definedabove, nucleic acid strands. In general, the majority of nucleotides ofeach strand are ribonucleotides, but as described in detail herein, eachor both strands can also include at least one non-ribonucleotide, e.g.,a deoxyribonucleotide and/or a modified nucleotide. In addition, as usedin this specification, “dsRNA” may include chemical modifications toribonucleotides, including substantial modifications at multiplenucleotides and including all types of modifications disclosed herein orknown in the art. Any such modifications, as used in an siRNA typemolecule, are encompassed by “dsRNA” for the purposes of thisspecification and claims

The two strands forming the duplex structure may be different portionsof one larger RNA molecule, or they may be separate RNA molecules. Wherethe two strands are part of one larger molecule, and therefore areconnected by an uninterrupted chain of nucleotides between the 3′-end ofone strand and the 5′-end of the respective other strand forming theduplex structure, the connecting RNA chain is referred to as a “hairpinloop.” Where the two strands are connected covalently by means otherthan an uninterrupted chain of nucleotides between the 3′-end of onestrand and the 5′-end of the respective other strand forming the duplexstructure, the connecting structure is referred to as a “linker.” TheRNA strands may have the same or a different number of nucleotides. Themaximum number of base pairs is the number of nucleotides in theshortest strand of the dsRNA minus any overhangs that are present in theduplex. In addition to the duplex structure, a dsRNA may comprise one ormore nucleotide overhangs. The term “siRNA” is also used herein to referto a dsRNA as described above.

As used herein, a “nucleotide overhang” refers to the unpairednucleotide or nucleotides that protrude from the duplex structure of adsRNA when a 3′-end of one strand of the dsRNA extends beyond the 5′-endof the other strand, or vice versa. “Blunt” or “blunt end” means thatthere are no unpaired nucleotides at that end of the dsRNA, i.e., nonucleotide overhang. A “blunt ended” dsRNA is a dsRNA that isdouble-stranded over its entire length, i.e., no nucleotide overhang ateither end of the molecule.

The term “antisense strand” refers to the strand of a dsRNA whichincludes a region that is substantially complementary to a targetsequence. As used herein, the term “region of complementarity” refers tothe region on the antisense strand that is substantially complementaryto a sequence, for example a target sequence, as defined herein. Wherethe region of complementarity is not fully complementary to the targetsequence, the mismatches are most tolerated in the terminal regions and,if present, are generally in a terminal region or regions, e.g., within6, 5, 4, 3, or 2 nucleotides of the 5′ and/or 3′ terminus.

The term “sense strand,” as used herein, refers to the strand of a dsRNAthat includes a region that is substantially complementary to a regionof the antisense strand.

As used herein, the term “SNALP” refers to a stable nucleic acid-lipidparticle. A SNALP represents a vesicle of lipids coating a reducedaqueous interior comprising a nucleic acid such as an iRNA agent or aplasmid from which an iRNA agent is transcribed. SNALP are described,e.g., in U.S. Patent Application Publication Nos. 20060240093,20070135372, and U.S. Ser. No. 61/045,228 filed on Apr. 15, 2008. Theseapplications are hereby incorporated by reference.

“Introducing into a cell,” when referring to a dsRNA, means facilitatinguptake or absorption into the cell, as is understood by those skilled inthe art. Absorption or uptake of dsRNA can occur through unaideddiffusive or active cellular processes, or by auxiliary agents ordevices. The meaning of this term is not limited to cells in vitro; adsRNA may also be “introduced into a cell,” wherein the cell is part ofa living organism. In such instance, introduction into the cell willinclude the delivery to the organism. For example, for in vivo delivery,dsRNA can be injected into a tissue site or administered systemically.In vitro introduction into a cell includes methods known in the art suchas electroporation and lipofection.

The terms “silence,” “inhibit the expression of,” “down-regulate theexpression of,” “suppress the expression of” and the like, in as far asthey refer to a target gene, herein refer to the at least partialsuppression of the expression of a GNAQ gene, as manifested by areduction of the amount of mRNA which may be isolated or detected from afirst cell or group of cells in which a GNAQ gene is transcribed andwhich has or have been treated such that the expression of a GNAQ geneis inhibited, as compared to a second cell or group of cellssubstantially identical to the first cell or group of cells but whichhas or have not been so treated (control cells). The degree ofinhibition is usually expressed in terms of

${\frac{\left( {{mRNA}\mspace{14mu}{in}\mspace{14mu}{control}\mspace{14mu}{cells}} \right) - \left( {{mRNA}\mspace{14mu}{in}\mspace{14mu}{treated}\mspace{14mu}{cells}} \right)}{\left( {{mRNA}\mspace{14mu}{in}\mspace{14mu}{control}\mspace{14mu}{cells}} \right)} \cdot 100}\%$

Alternatively, the degree of inhibition may be given in terms of areduction of a parameter that is functionally linked to GNAQ genetranscription, e.g., the amount of protein encoded by a GNAQ gene whichis secreted by a cell, or the number of cells displaying a certainphenotype, e.g., apoptosis. In principle, GNAQ gene silencing may bedetermined in any cell expressing the target, either constitutively orby genomic engineering, and by any appropriate assay. However, when areference is needed in order to determine whether a given dsRNA inhibitsthe expression of a GNAQ gene by a certain degree and therefore isencompassed by the instant invention, the assays provided in theExamples below shall serve as such reference.

For example, in certain instances, expression of a GNAQ gene issuppressed by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,or 50% by administration of the double-stranded oligonucleotide featuredin the invention. In some embodiments, a GNAQ gene is suppressed by atleast about 60%, 70%, or 80% by administration of the double-strandedoligonucleotide featured in the invention. In some embodiments, a GNAQgene is suppressed by at least about 85%, 90%, or 95% by administrationof the double-stranded oligonucleotide featured in the invention.

As used herein in the context of GNAQ expression, the terms “treat,”“treatment,” and the like, refer to relief from or alleviation ofpathological processes mediated by GNAQ expression. In the context ofthe present invention insofar as it relates to any of the otherconditions recited herein below (other than pathological processesmediated by GNAQ expression), the terms “treat,” “treatment,” and thelike mean to relieve or alleviate at least one symptom associated withsuch condition, or to slow or reverse the progression of such condition,such as tumor reduction in uveal melanoma.

As used herein, the phrases “therapeutically effective amount” and“prophylactically effective amount” refer to an amount that provides atherapeutic benefit in the treatment, prevention, or management ofpathological processes mediated by GNAQ expression or an overt symptomof pathological processes mediated by GNAQ expression. The specificamount that is therapeutically effective can be readily determined by anordinary medical practitioner, and may vary depending on factors knownin the art, such as, for example, the type of pathological processesmediated by GNAQ expression, the patient's history and age, the stage ofpathological processes mediated by GNAQ expression, and theadministration of other anti-pathological processes mediated by GNAQexpression agents.

As used herein, a “pharmaceutical composition” comprises apharmacologically effective amount of a dsRNA and a pharmaceuticallyacceptable carrier. As used herein, “pharmacologically effectiveamount,” “therapeutically effective amount” or simply “effective amount”refers to that amount of an RNA effective to produce the intendedpharmacological, therapeutic or preventive result. For example, if agiven clinical treatment is considered effective when there is at leasta 25% reduction in a measurable parameter associated with a disease ordisorder, a therapeutically effective amount of a drug for the treatmentof that disease or disorder is the amount necessary to effect at least a25% reduction in that parameter.

The term “pharmaceutically acceptable carrier” refers to a carrier foradministration of a therapeutic agent. Such carriers include, but arenot limited to, saline, buffered saline, dextrose, water, glycerol,ethanol, and combinations thereof. The term specifically excludes cellculture medium. For drugs administered orally, pharmaceuticallyacceptable carriers include, but are not limited to pharmaceuticallyacceptable excipients such as inert diluents, disintegrating agents,binding agents, lubricating agents, sweetening agents, flavoring agents,coloring agents and preservatives. Suitable inert diluents includesodium and calcium carbonate, sodium and calcium phosphate, and lactose,while corn starch and alginic acid are suitable disintegrating agents.Binding agents may include starch and gelatin, while the lubricatingagent, if present, will generally be magnesium stearate, stearic acid ortalc. If desired, the tablets may be coated with a material such asglyceryl monostearate or glyceryl distearate, to delay absorption in thegastrointestinal tract.

As used herein, a “transformed cell” is a cell into which a vector hasbeen introduced from which a dsRNA molecule may be expressed.

Double-stranded Ribonucleic Acid (dsRNA)

As described in more detail herein, the invention providesdouble-stranded ribonucleic acid (dsRNA) molecules for inhibiting theexpression of a GNAQ gene in a cell or mammal, where the dsRNA includesa sense strand having a first sequence and an antisense strandcomprising a second sequence complementary to mRNA encoding GNAQ,wherein said first sequence is complementary to said second sequence ata region of complementarity and wherein each strand is 15 to 30 basepairs in length. In some embodiments, the dsRNA of the inventioninhibits the expression of said GNAQ gene by at least 40% as assayed by,for example, a PCR or branched DNA (bDNA)-based method, or by aprotein-based method, such as by Western blot. Expression of a GNAQ genecan be reduced by at least 30% when measured by an assay as described inthe Examples below. For example, expression of a GNAQ gene in cellculture, such as in HepB3 cells, can be assayed by measuring GNAQ mRNAlevels, such as by bDNA or TaqMan assay, or by measuring protein levels,such as by ELISA assay.

The dsRNA can be synthesized by standard methods known in the art asfurther discussed below, e.g., by use of an automated DNA synthesizer,such as are commercially available from, for example, Biosearch, AppliedBiosystems, Inc. The dsRNA includes two RNA strands that aresufficiently complementary to hybridize to form a duplex structure.

One strand of the dsRNA (the antisense strand) includes a region ofcomplementarity that is complementary, to a target sequence, derivedfrom the sequence of an mRNA formed during the expression of a targetgene, the other strand (the sense strand) includes a region that iscomplementary to the antisense strand, such that the two strandshybridize and form a duplex structure when combined under suitableconditions. The region of complementarity is generally at least 15nucleotides in length, or between 19 and 21 nucleotides in length, or19, 20, or 21 nucleotides in length. In some embodiments the region ofcomplementarity includes at least 15 contiguous nucleotides of one ofthe antisense sequences listed in Tables 2a, 3a, or 4a. In otherembodiments the region of complementarity includes one of the antisensesequences listed in Tables 2a, 3a, or 4a.

Generally, the duplex structure is between 15 and 30, or between 25 and30, or between 18 and 25, or between 19 and 24, or between 19 and 21, or19, 20, or 21 base pairs in length. In one embodiment the duplex is 19base pairs in length. In another embodiment the duplex is 21 base pairsin length. When two different dsRNAs are used in combination, the duplexlengths can be identical or can differ.

Each strand of the dsRNA of invention is generally between 15 and 30, orbetween 18 and 25, or 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides inlength. In other embodiments, each is strand is 25-30 nucleotides inlength. Each strand of the duplex can be the same length or of differentlengths. When two different siRNAs are used in combination, the lengthsof each strand of each siRNA can be identical or can differ.

The dsRNA of the invention can include one or more single-strandedoverhang(s) of one or more nucleotides. In one embodiment, at least oneend of the dsRNA has a single-stranded nucleotide overhang of 1 to 4, or1, 2, 3, or 4 nucleotides. In another embodiment, the overhang includedTdT. In another embodiment, the antisense strand of the dsRNA has 1-10nucleotides overhangs each at the 3′ end and the 5′ end over the sensestrand. In further embodiments, the sense strand of the dsRNA has 1-10nucleotides overhangs each at the 3′ end and the 5′ end over theantisense strand.

A dsRNAs having at least one nucleotide overhang can have unexpectedlysuperior inhibitory properties than the blunt-ended counterpart. In someembodiments the presence of only one nucleotide overhang strengthens theinterference activity of the dsRNA, without affecting its overallstability. A dsRNA having only one overhang has proven particularlystable and effective in vivo, as well as in a variety of cells, cellculture mediums, blood, and serum. Generally, the single-strandedoverhang is located at the 3′-terminal end of the antisense strand or,alternatively, at the 3′-terminal end of the sense strand. The dsRNA canalso have a blunt end, generally located at the 5′-end of the antisensestrand. Such dsRNAs can have improved stability and inhibitory activity,thus allowing administration at low dosages, i.e., less than 5 mg/kgbody weight of the recipient per day. Generally, the antisense strand ofthe dsRNA has a nucleotide overhang at the 3′-end, and the 5′-end isblunt. In another embodiment, one or more of the nucleotides in theoverhang is replaced with a nucleoside thiophosphate.

In one embodiment, a GNAQ gene is a human GNAQ gene, e.g., the sequenceidentified by GenBank accession number NM_002072.2.

In specific embodiments, the sense strand of the dsRNA is one of the asense sequences from Tables 2-4, and the antisense strand is one of theantisense sequences of Tables 2-4. Alternative antisense agents thattarget elsewhere in the target sequence provided in Tables 2-4 canreadily be determined using the target sequence and the flanking GNAQsequence.

The skilled person is well aware that dsRNAs having a duplex structureof between 20 and 23, but specifically 21, base pairs have been hailedas particularly effective in inducing RNA interference (Elbashir et al.,EMBO 2001, 20:6877-6888). However, others have found that shorter orlonger dsRNAs can be effective as well. In the embodiments describedabove, by virtue of the nature of the oligonucleotide sequences providedin Tables 2-4, the dsRNAs featured in the invention can include at leastone strand of a length described therein. It can be reasonably expectedthat shorter dsRNAs having one of the sequences of Tables 2-4 minus onlya few nucleotides on one or both ends may be similarly effective ascompared to the dsRNAs described above. Hence, dsRNAs having a partialsequence of at least 15, 16, 17, 18, 19, 20, 21, or 22, or morecontiguous nucleotides from one of the sequences of Tables 2-4, anddiffering in their ability to inhibit the expression of a GNAQ gene inan assay as described herein below by not more than 5, 10, 15, 20, 25,or 30% inhibition from a dsRNA comprising the full sequence, arecontemplated by the invention. Further, dsRNAs that cleave within adesired GNAQ target sequence can readily be made using the correspondingGNAQ antisense sequence and a complementary sense sequence.

In addition, the dsRNAs provided in Tables 2-4 identify a site in a GNAQthat is susceptible to RNAi based cleavage. As such, the presentinvention further features dsRNAs that target within the sequencetargeted by one of the agents of the present invention. As used herein,a second dsRNA is said to target within the sequence of a first dsRNA ifthe second dsRNA cleaves the message anywhere within the mRNA that iscomplementary to the antisense strand of the first dsRNA. Such a seconddsRNA will generally consist of at least 15 contiguous nucleotides fromone of the sequences provided in Tables 2-4 coupled to additionalnucleotide sequences taken from the region contiguous to the selectedsequence in a GNAQ gene.

Additional dsRNA of the invention include those that cleave a targetmRNA at the same location as a dsRNA described in any of the tables. Ingeneral, a RISC complex will cleave a target mRNA between thenucleotides complementary to nucleotides 10 and 11 of the antisensestrand of a dsRNA, e.g., siRNA, of the invention. Cleavage e sites canbe assayed using, e.g., a 5′ RACE assay.

For example, the duplex AD-20057 includes the sense and antisensestrands below. Treatment of a cell with this duplex results in cleavageof human GNAQ mRNA at the nucleotides complementary to nucleotides 10and 11 of the antisense strand, e.g., nucleotides 1211 and 1212.Therefore, also included in the invention are those dsRNA that cleave atthat location.

The dsRNA featured in the invention can contain one or more mismatchesto the target sequence. In one embodiment, the dsRNA featured in theinvention contains no more than 3 mismatches. If the antisense strand ofthe dsRNA contains mismatches to a target sequence, it is preferablethat the area of mismatch not be located in the center of the region ofcomplementarity. If the antisense strand of the dsRNA containsmismatches to the target sequence, it is preferable that the mismatch berestricted to 5 nucleotides from either end, for example 5, 4, 3, 2, or1 nucleotide from either the 5′ or 3′ end of the region ofcomplementarity. For example, for a 23 nucleotide dsRNA strand which iscomplementary to a region of a target gene, the dsRNA generally does notcontain any mismatch within the central 13 nucleotides. The methodsdescribed within the invention can be used to determine whether a dsRNAcontaining a mismatch to a target sequence is effective in inhibitingthe expression of a target gene. Consideration of the efficacy of dsRNAswith mismatches in inhibiting expression of a target gene is important,especially if the particular region of complementarity in a target geneis known to have polymorphic sequence variation within the population.

Modifications

In yet another embodiment, the dsRNA is chemically modified to enhancestability. The nucleic acids featured in the invention may besynthesized and/or modified by methods well established in the art, suchas those described in “Current protocols in nucleic acid chemistry,”Beaucage, S. L. et al. (Edrs.), John Wiley & Sons, Inc., New York, N.Y.,USA, which is hereby incorporated herein by reference. Specific examplesof dsRNA compounds useful in this invention include dsRNAs containingmodified backbones or no natural internucleoside linkages. As defined inthis specification, dsRNAs having modified backbones include those thatretain a phosphorus atom in the backbone and those that do not have aphosphorus atom in the backbone. For the purposes of this specification,and as sometimes referenced in the art, modified dsRNAs that do not havea phosphorus atom in their internucleoside backbone can also beconsidered to be oligonucleosides.

Modified dsRNA backbones include, for example, phosphorothioates, chiralphosphorothioates, phosphorodithioates, phosphotriesters,aminoalkylphosphotriesters, methyl and other alkyl phosphonatesincluding 3′-alkylene phosphonates and chiral phosphonates,phosphinates, phosphoramidates including 3′-amino phosphoramidate andaminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, andboranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs ofthese, and those) having inverted polarity wherein the adjacent pairs ofnucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Varioussalts, mixed salts and free acid forms are also included.

Representative U.S. patents that teach the preparation of the abovephosphorus-containing linkages include, but are not limited to, U.S.Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,195;5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131;5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925;5,519,126; 5,536,821; 5,541,316; 5,550,111; 5,563,253; 5,571,799;5,587,361; and 5,625,050, each of which is herein incorporated byreference

Modified dsRNA backbones that do not include a phosphorus atom thereinhave backbones that are formed by short chain alkyl or cycloalkylinternucleoside linkages, mixed heteroatoms and alkyl or cycloalkylinternucleoside linkages, or ore or more short chain heteroatomic orheterocyclic internucleoside linkages. These include those havingmorpholino linkages (formed in part from the sugar portion of anucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; alkene containing backbones; sulfamatebackbones; methyleneimino and methylenehydrazino backbones; sulfonateand sulfonamide backbones; amide backbones; and others having mixed N,O, S and CH2 component parts.

Representative U.S. patents that teach the preparation of the aboveoligonucleosides include, but are not limited to, U.S. Pat. Nos.5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033;5,64,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,608,046;5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and,5,677,439, each of which is herein incorporated by reference.

In other suitable dsRNA mimetics, both the sugar and the internucleosidelinkage, i.e., the backbone, of the nucleotide units are replaced withnovel groups. The base units are maintained for hybridization with anappropriate nucleic acid target compound. One such oligomeric compound,a dsRNA mimetic that has been shown to have excellent hybridizationproperties, is referred to as a peptide nucleic acid (PNA). In PNAcompounds, the sugar backbone of a dsRNA is replaced with an amidecontaining backbone, in particular an aminoethylglycine backbone. Thenucleobases are retained and are bound directly or indirectly to azanitrogen atoms of the amide portion of the backbone. Representative U.S.patents that teach the preparation of PNA compounds include, but are notlimited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each ofwhich is herein incorporated by reference. Further teaching of PNAcompounds can be found in Nielsen et al., Science, 1991, 254, 1497-1500.

Other embodiments of the invention are dsRNAs with phosphorothioatebackbones and oligonucleosides with heteroatom backbones, and inparticular —CH2—NH—CH2—, —CH2—N(CH3)—O—CH2—[known as a methylene(methylimino) or MMI backbone], —CH2—O—N(CH3)—CH2—,—CH2—N(CH3)—N(CH3)—CH2— and —N(CH3)—CH2—CH2—[wherein the nativephosphodiester backbone is represented as —O—P—O—CH2—] of theabove-referenced U.S. Pat. No. 5,489,677, and the amide backbones of theabove-referenced U.S. Pat. No. 5,602,240. Also preferred are dsRNAshaving morpholino backbone structures of the above-referenced U.S. Pat.No. 5,034,506.

Modified dsRNAs may also contain one or more substituted sugar moieties.Preferred dsRNAs comprise one of the following at the 2′ position: OH;F; O—, S—, or N-alkyl; O—, S—, or N-alkenyl; O—, S—or N-alkynyl; orO-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may besubstituted or unsubstituted C1 to C10 alkyl or C2 to C10 alkenyl andalkynyl. Particularly preferred are O[(CH2)nO]mCH3, O(CH2)nOCH3,O(CH2)nNH2, O(CH2)nCH3, O(CH2)nONH2, and O(CH2)nON[(CH2)nCH3)]2, where nand m are from 1 to about 10. Other preferred dsRNAs comprise one of thefollowing at the 2′ position: C1 to C10 lower alkyl, substituted loweralkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br,CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl,heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl,an RNA cleaving group, a reporter group, an intercalator, a group forimproving the pharmacokinetic properties of an dsRNA, or a group forimproving the pharmacodynamic properties of an dsRNA, and othersubstituents having similar properties. A preferred modificationincludes 2′-methoxyethoxy (2′-O—CH2CH2OCH3, also known as2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv. Chim. Acta, 1995,78, 486-504) i.e., an alkoxy-alkoxy group. A further preferredmodification includes 2′-dimethylaminooxyethoxy, i.e., a O(CH2)2ON(CH3)2group, also known as 2′-DMAOE, as described in examples herein below,and 2′-dimethylaminoethoxyethoxy (also known in the art as2′-O-dimethylaminoethoxyethyl or 2′-DMAEOE), i.e.,2′-O—CH2—O—CH2—N(CH2)2, also described in examples herein below.

Other preferred modifications include 2′-methoxy (2′-OCH₃),2′-aminopropoxy (2′-OCH₂CH₂CH₂NH₂) and 2′-fluoro (2′-F). Similarmodifications may also be made at other positions on the dsRNA,particularly the 3′ position of the sugar on the 3′ terminal nucleotideor in 2′-5′ linked dsRNAs and the 5′ position of 5′ terminal nucleotide.DsRNAs may also have sugar mimetics such as cyclobutyl moieties in placeof the pentofuranosyl sugar. Representative U.S. patents that teach thepreparation of such modified sugar structures include, but are notlimited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044;5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811;5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873;5,646,265; 5,658,873; 5,670,633; and 5,700,920, certain of which arecommonly owned with the instant application, and each of which is hereinincorporated by reference in its entirety.

A dsRNA may also include nucleobase (often referred to in the art simplyas “base”) modifications or substitutions. As used herein, “unmodified”or “natural” nucleobases include the purine bases adenine (A) andguanine (G), and the pyrimidine bases thymine (T), cytosine (C) anduracil (U). Modified nucleobases include other synthetic and naturalnucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine,xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkylderivatives of adenine and guanine, 2-propyl and other alkyl derivativesof adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine,5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil,cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo,8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl anal other 8-substitutedadenines and guanines, 5-halo, particularly 5-bromo, 5-trifluoromethyland other 5-substituted uracils and cytosines, 7-methylguanine and7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and7-daazaadenine and 3-deazaguanine and 3-deazaadenine. Furthernucleobases include those disclosed in U.S. Pat. No. 3,687,808, thosedisclosed in The Concise Encyclopedia Of Polymer Science AndEngineering, pages 858-859, Kroschwitz, J. L, ed. John Wiley & Sons,1990, these disclosed by Englisch et al., Angewandte Chemie,International Edition, 1991, 30, 613, and those disclosed by Sanghvi, YS., Chapter 15, DsRNA Research and Applications, pages 289-302, Crooke,S. T. and Lebleu, B., Ed., CRC Press, 1993. Certain of these nucleobasesare particularly useful for increasing the binding affinity of theoligomeric compounds featured in the invention. These include5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6substituted purines, including 2-aminopropyladenine, 5-propynyluraciland 5-propynylcytosine. 5-methylcytosine substitutions have been shownto increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y.S., Crooke, S. T. and Lebleu, B., Eds., DsRNA Research and Applications,CRC Press, Boca Raton, 1993, pp. 276-278) and are exemplary basesubstitutions, even more particularly when combined with2′-O-methoxyethyl sugar modifications.

Representative U.S. patents that teach the preparation of certain of theabove noted modified nucleobases as well as other modified nucleobasesinclude, but are not limited to, the above noted U.S. Pat. No.3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,30; 5,134,066;5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908;5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091;5,614,617; and 5,681,941, each of which is herein incorporated byreference, and U.S. Pat. No. 5,750,692, also herein incorporated byreference.

Conjugates

Another modification of the dsRNAs featured in the invention involveschemically linking to the dsRNA one or more moieties or conjugates whichenhance the activity, cellular distribution or cellular uptake of thedsRNA. Such moieties include but are not limited to lipid moieties suchas a cholesterol moiety (Letsinger et al., Proc. Natl. Acid. Sci. USA,1989, 86: 6553-6556), cholic acid (Manoharan et al., Biorg. Med. Chem.Let., 1994, 4:1053-1060), a thioether, e.g., beryl-S-tritylthiol(Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660:306-309; Manoharan etal., Biorg. Med. Chem. Let., 1993, 3:2765-2770), a thiocholesterol(Oberhauser et al., Nucl. Acids Res., 1992, 20:533-538), an aliphaticchain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al.,EMBO J, 1991, 10:1111-1118; Kabanov et al., FEBS Lett., 1990,259:327-330; Svinarchuk et al., Biochimie, 1993, 75:49-54), aphospholipid, e.g., di-hexadecyl-rac-glycerol or triethyl-ammonium1,2-di-O-hexadecyl-rac-glycero-3-Hphosphonate (Manoharan et al.,Tetrahedron Lett., 1995, 36:3651-3654; Shea et al., Nucl. Acids Res.,1990, 18:3777-3783), a polyamine or a polyethylene glycol chain(Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969-973), oradamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995,36:3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta,1995, 1264:229-237), or an octadecylamine orhexylamino-carbonyloxycholesterol moiety (Crooke et al., J. Pharmacol.Exp. Ther., 1996, 277:923-937).

Representative U.S. patents that teach the preparation of such dsRNAconjugates include, but are not limited to, U.S. Pat. Nos. 4,828,979;4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538;5,578,717, 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045;5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044;4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263;4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136;5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506;5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723;5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552;5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696;5,599,923; 5,599,928 and 5,688,941, each of which is herein incorporatedby reference.

It is not necessary for all positions in a given compound to beuniformly modified, and in fact more than one of the aforementionedmodifications may be incorporated in a single compound or even at asingle nucleoside within a dsRNA. The present invention also includesdsRNA compounds which are chimeric compounds. “Chimeric” dsRNA compoundsor “chimeras,” in the context of this invention, are dsRNA compounds,particularly dsRNAs, which contain two or more chemically distinctregions, each made up of at least one monomer unit, i.e., a nucleotidein the case of a dsRNA compound. These dsRNAs typically contain at leastone region wherein the dsRNA is modified so as to confer upon the dsRNAincreased resistance to nuclease degradation, increased cellular uptake,and/or increased binding affinity for the target nucleic acid. Anadditional region of the dsRNA may serve as a substrate for enzymescapable of cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNaseH is a cellular endonuclease which cleaves the RNA strand of an RNA:DNAduplex. Activation of RNase H, therefore, results in cleavage of the RNAtarget, thereby greatly enhancing the efficiency of dsRNA inhibition ofgene expression. Consequently, comparable results can often be obtainedwith shorter dsRNAs when chimeric dsRNAs are used, compared tophosphorothioate deoxydsRNAs hybridizing to the same target region.Cleavage of the RNA target can be routinely detected by gelelectrophoresis and, if necessary, associated nucleic acid hybridizationtechniques known in the art.

In certain instances, the dsRNA may be modified by a non-ligand group. Anumber of non-ligand molecules have been conjugated to dsRNAs in orderto enhance the activity, cellular distribution or cellular uptake of thedsRNA, and procedures for performing such conjugations are available inthe scientific literature. Such non-ligand moieties have included lipidmoieties, such as cholesterol (Letsinger et al., Proc. Natl. Acad. Sci.USA, 1989, 86:6553), cholic acid (Manoharan et al., Bioorg. Med. Chem.Lett., 1994, 4:1053), a thioether, e.g., hexyl-S-tritylthiol (Manoharanet al., Ann. N.Y. Acad. Sci., 1992, 660:306; Manoharan et al., Bioorg.Med. Chem. Let., 1993, 3:2765), a thiocholesterol (Oberhauser et al.,Nucl. Acids Res., 1992, 20:533), an aliphatic chain, e.g., dodecandiolor undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10:111;Kabanov et al., FEBS Left., 1990, 259:327; Svinarchuk et al., Biochimie,1993, 75:49), a phospholipid, e.g., di-hexadecyl-rac-glycerol ortriethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate(Manoharan et al., Tetrahedron Lett., 1995, 36:3651; Shea et al., Nucl.Acids Res., 1990, 18:3777), a polyamine or a polyethylene glycol chain(Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969), oradamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995,36:3651), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta,1995, 1264:229), or an octadecylamine orhexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol.Exp. Ther., 1996, 277:923). Representative United States patents thatteach the preparation of such dsRNA conjugates have been listed above.Typical conjugation protocols involve the synthesis of dsRNAs bearing anaminolinker at one or more positions of the sequence. The amino group isthen reacted with the molecule being conjugated using appropriatecoupling or activating reagents. The conjugation reaction may beperformed either with the dsRNA still bound to the solid support orfollowing cleavage of the dsRNA in solution phase. Purification of thedsRNA conjugate by HPLC typically affords the pure conjugate.

Vector Encoded dsRNAs

In another aspect, dsRNA molecules of the invention are expressed fromtranscription units inserted into DNA or RNA vectors (see, e.g.,Couture, A, et al., TIG. (1996), 12:5-10; Skillern, A., et al.,International PCT Publication No. WO 00/22113, Conrad, International PCTPublication No. WO 00/22114, and Conrad, U.S. Pat. No. 6,054,299). Thesetransgenes can be introduced as a linear construct, a circular plasmid,or a viral vector, which can be incorporated and inherited as atransgene integrated into the host genome. The transgene can also beconstructed to permit it to be inherited as an extrachromosomal plasmid(Gassmann, et al., Proc. Natl. Acad. Sci. USA (1995) 92:1292).

The individual strands of a dsRNA can be transcribed by promoters on twoseparate expression vectors and co-transfected into a target cell.Alternatively each individual strand of the dsRNA can be transcribed bypromoters both of which are located on the same expression plasmid. Inone embodiment, a dsRNA is expressed as an inverted repeat joined by alinker polynucleotide sequence such that the dsRNA has a stem and loopstructure.

The recombinant dsRNA expression vectors are generally DNA plasmids orviral vectors. dsRNA expressing viral vectors can be constructed basedon, but not limited to, adeno-associated virus (for a review, seeMuzyczka, et al., Curr. Topics Micro. Immunol. (1992) 158:97-129));adenovirus (see, for example, Berkner, et al., BioTechniques (1998)6:616), Rosenfeld et al. (1991, Science 252:431-434), and Rosenfeld etal. (1992), Cell 68:143-155)); or alphavirus as well as others known inthe art. Retroviruses have been used to introduce a variety of genesinto many different cell types, including epithelial cells, in vitroand/or in vivo (see, e.g., Eglitis, et al., Science (1985)230:1395-1398; Danos and Mulligan, Proc. NatI. Acad. Sci. USA (1998)85:6460-6464; Wilson et al., 1988, Proc. Natl. Acad. Sci. USA85:3014-3018; Armentano et al., 1990, Proc. Natl. Acad. Sci. USA87:61416145; Huber et al., 1991, Proc. Natl. Acad. Sci. USA88:8039-8043; Ferry et al., 1991, Proc. Natl. Acad. Sci. USA88:8377-8381; Chowdhury et al., 1991, Science 254:1802-1805; vanBeusechem. et al., 1992, Proc. Nad. Acad. Sci. USA 89:7640-19; Kay etal., 1992, Human Gene Therapy 3:641-647; Dai et al., 1992, Proc. Natl.Acad. Sci. USA 89:10892-10895; Hwu et al., 1993, J. Immunol.150:4104-4115; U.S. Pat. No. 4,868,116; U.S. Pat. No. 4,980,286; PCTApplication WO 89/07136; PCT Application WO 89/02468; PCT Application WO89/05345; and PCT Application WO 92/07573). Recombinant retroviralvectors capable of transducing and expressing genes inserted into thegenome of a cell can be produced by transfecting the recombinantretroviral genome into suitable packaging cell lines such as PA317 andPsi-CRIP (Comette et al., 1991, Human Gene Therapy 2:5-10; Cone et al.,1984, Proc. Natl. Acad. Sci. USA 81:6349). Recombinant adenoviralvectors can be used to infect a wide variety of cells and tissues insusceptible hosts (e.g., rat, hamster, dog, and chimpanzee) (Hsu et al.,1992, J. Infectious Disease, 166:769), and also have the advantage ofnot requiring mitotically active cells for infection.

Any viral vector capable of accepting the coding sequences for the dsRNAmolecule(s) to be expressed can be used, for example vectors derivedfrom adenovirus (AV); adeno-associated virus (AAV); retroviruses (e.g.,lentiviruses (LV), Rhabdoviruses, murine leukemia virus); herpes virus,and the like. The tropism of viral vectors can be modified bypseudotyping the vectors with envelope proteins or other surfaceantigens from other viruses, or by substituting different viral capsidproteins, as appropriate.

For example, lentiviral vectors featured in the invention can bepseudotyped with surface proteins from vesicular stomatitis virus (VSV),rabies, Ebola, Mokola, and the like. AAV vectors featured in theinvention can be made to target different cells by engineering thevectors to express different capsid protein serotypes. For example, anAAV vector expressing a serotype 2 capsid on a serotype 2 genome iscalled AAV 2/2. This serotype 2 capsid gene in the AAV 2/2 vector can bereplaced by a serotype 5 capsid gene to produce an AAV 2/5 vector.Techniques for constructing AAV vectors which express different capsidprotein serotypes are within the skill in the art; see, e.g., RabinowitzJ E et al. (2002), J Virol 76:791-801, the entire disclosure of which isherein incorporated by reference.

Selection of recombinant viral vectors suitable for use in theinvention, methods for inserting nucleic acid sequences for expressingthe dsRNA into the vector, and methods of delivering the viral vector tothe cells of interest are within the skill in the art. See, for example,Dornburg R (1995), Gene Therap. 2: 301-310; Eglitis M A (1988),Biotechniques 6: 608-614; Miller A D (1990), Hum Gene Therap. 1: 5-14;Anderson W F (1998), Nature 392: 25-30; and Rubinson D A et al., Nat.Genet. 33: 401-406, the entire disclosures of which are hereinincorporated by reference.

Viral vectors can be derived from AV and AAV. In one embodiment, thedsRNA featured in the invention is expressed as two separate,complementary single-stranded RNA molecules from a recombinant AAVvector having, for example, either the U6 or H1 RNA promoters, or thecytomegalovirus (CMV) promoter.

A suitable AV vector for expressing the dsRNA featured in the invention,a method for constructing the recombinant AV vector, and a method fordelivering the vector into target cells, are described in Xia H et al.(2002), Nat. Biotech. 20: 1006-1010.

Suitable AAV vectors for expressing the dsRNA featured in the invention,methods for constructing the recombinant AV vector, and methods fordelivering the vectors into target cells are described in Samulski R etal. (1987), J. Virol. 61: 3096-3101; Fisher K J et al. (1996), J. Virol,70: 520-532; Samulski R et al. (1989), J. Virol. 63: 3822-3826; U.S.Pat. No. 5,252,479; U.S. Pat. No. 5,139,941; International PatentApplication No. WO 94/13788; and International Patent Application No. WO93/24641, the entire disclosures of which are herein incorporated byreference.

The promoter driving dsRNA expression in either a DNA plasmid or viralvector featured in the invention may be a eukaryotic RNA polymerase I(e.g., ribosomal RNA promoter), RNA polymerase II (e.g., CMV earlypromoter or actin promoter or U1 snRNA promoter) or generally RNApolymerase III promoter (e.g., U6 snRNA or 7SK RNA promoter) or aprokaryotic promoter, for example the T7 promoter, provided theexpression plasmid also encodes T7 RNA polymerase required fortranscription from a T7 promoter. The promoter can also direct transgeneexpression to the pancreas (see, e.g., the insulin regulatory sequencefor pancreas (Bucchini et al., 1986, Proc. Natl. Acad. Sci. USA83:2511-2515)).

In addition, expression of the transgene can be precisely regulated, forexample, by using an inducible regulatory sequence and expressionsystems such as a regulatory sequence that is sensitive to certainphysiological regulators, e.g., circulating glucose levels, or hormones(Docherty et al., 1994, FASEB J. 8:20-24). Such inducible expressionsystems, suitable for the control of transgene expression in cells or inmammals include regulation by ecdysone, by estrogen, progesterone,tetracycline, chemical inducers of dimerization, andisopropyl-beta-D1-thiogalactopyranoside (EPTG). A person skilled in theart would be able to choose the appropriate regulatory/promoter sequencebased on the intended use of the dsRNA transgene.

Generally, recombinant vectors capable of expressing dsRNA molecules aredelivered as described below, and persist in target cells.Alternatively, viral vectors can be used that provide for transientexpression of dsRNA molecules. Such vectors can be repeatedlyadministered as necessary. Once expressed, the dsRNAs bind to target RNAand modulate its function or expression. Delivery of dsRNA expressingvectors can be systemic, such as by intravenous or intramuscularadministration, by administration to target cells ex-planted from thepatient followed by reintroduction into the patient, or by any othermeans that allows for introduction into a desired target cell.

dsRNA expression DNA plasmids are typically transfected into targetcells as a complex with cationic lipid carriers (e.g., Oligofectamine)or non-cationic lipid-based carriers (e.g., Transit-TKO™). Multiplelipid transfections for dsRNA-mediated knockdowns targeting differentregions of a single target gene or multiple target genes over a periodof a week or more are also contemplated by the invention. Successfulintroduction of vectors into host cells can be monitored using variousknown methods. For example, transient transfection can be signaled witha reporter, such as a fluorescent marker, such as Green FluorescentProtein (GFP). Stable transfection of cells ex vivo can be ensured usingmarkers that provide the transfected cell with resistance to specificenvironmental factors (e.g., antibiotics and drugs), such as hygromycinB resistance.

Target gene specific dsRNA molecules can also be inserted into vectorsand used as gene therapy vectors for human patients. Gene therapyvectors can be delivered to a subject by, for example, intravenousinjection, local administration (see U.S. Pat. No. 5,328,470) or bystereotactic injection (see e.g., Chen et al. (1994) Proc. Natl. Acad.Sci. USA 91:3054-3057). The pharmaceutical preparation of the genetherapy vector can include the gene therapy vector in an acceptablediluent, or can include a slow release matrix in which the gene deliveryvehicle is imbedded. Alternatively, where the complete gene deliveryvector can be produced intact from recombinant cells, e.g., retroviralvectors, the pharmaceutical preparation can include one or more cellswhich produce the gene delivery system.

Pharmaceutical Compositions Containing dsRNA

In one embodiment, the invention provides pharmaceutical compositionscontaining a dsRNA, as described herein, and a pharmaceuticallyacceptable carrier. The pharmaceutical composition containing the dsRNAis useful for treating a disease or disorder associated with theexpression or activity of a GNAQ gene, such as pathological processesmediated by GNAQ expression. Such pharmaceutical compositions areformulated based on the mode of delivery. One example is compositionsthat are formulated for systemic administration via parenteral delivery,e.g., by intravenous (IV) delivery. Another example is compositions thatare formulated for direct delivery into the brain parenchyma, e.g., byinfusion into the brain, such as by continuous pump infusion.

The pharmaceutical compositions featured herein are administered indosages sufficient to inhibit expression of GNAQ genes. In general, asuitable dose of dsRNA will be in the range of 0.01 to 200.0 milligramssiRNA per kilogram body weight of the recipient per day, generally inthe range of 1 to 50 mg per kilogram body weight per day. For example,the dsRNA can be administered at 0.0059 mg/kg, 0.01 mg/kg, 0.0295 mg/kg,0.05 mg/kg, 0.0590 mg/kg, 0.163 mg/kg, 0.2 mg/kg, 0.3 mg/kg, 0.4 mg/kg,0.5 mg/kg, 0.543 mg/kg, 0.5900 mg/kg, 0.6 mg/kg, 0.7 mg/kg, 0.8 mg/kg,0.9 mg/kg, 1 mg/kg, 1.1 mg/kg, 1.2 mg/kg, 1.3 mg/kg, 1.4 mg/kg, 1.5mg/kg, 1.628 mg/kg, 2 mg/kg, 3 mg/kg, 5.0 mg/kg, 10 mg/kg, 20 mg/kg, 30mg/kg, 40 mg/kg, or 50 mg/kg per single dose.

In one embodiment, the dosage is between 0.01 and 0.2 mg/kg. Forexample, the dsRNA can be administered at a dose of 0.01 mg/kg, 0.02mg/kg, 0.03 mg/kg, 0.04 mg/kg, 0.05 mg/kg, 0.06 mg/kg, 0.07 mg/kg 0.08mg/kg 0.09 mg/kg, 0.10 mg/kg, 0.11 mg/kg, 0.12 mg/kg, 0.13 mg/kg, 0.14mg/kg, 0.15 mg/kg, 0.16 mg/kg, 0.17 mg/kg, 0.18 mg/kg, 0.19 mg/kg, or0.20 mg/kg.

In one embodiment, the dosage is between 0.005 mg/kg and 1.628 mg/kg.For example, the dsRNA can be administered at a dose of 0.0059 mg/kg,0.0295 mg/kg, 0.0590 mg/kg, 0.163 mg/kg, 0.543 mg/kg, 0.5900 mg/kg, or1.628 mg/kg.

In one embodiment, the dosage is between 0.2 mg/kg and 1.5 mg/kg. Forexample, the dsRNA can be administered at a dose of 0.2 mg/kg, 0.3mg/kg, 0.4 mg/kg, 0.5 mg/kg, 0.6 mg/kg, 0.7 mg/kg, 0.8 mg/kg, 0.9 mg/kg,1 mg/kg, 1.1 mg/kg, 1.2 mg/kg, 1.3 mg/kg, 1.4 mg/kg, or 1.5 mg/kg.

The dsRNA can be administered at a dose of 0.03 mg/kg.

The pharmaceutical composition may be administered once daily, or thedsRNA may be administered as two, three, or more sub-doses atappropriate intervals throughout the day or even using continuousinfusion or delivery through a controlled release formulation. In thatcase, the dsRNA contained in each sub-dose must be correspondinglysmaller in order to achieve the total daily dosage. The dosage unit canalso be compounded for delivery over several days, e.g., using aconventional sustained release formulation which provides sustainedrelease of the dsRNA over a several day period. Sustained releaseformulations are well known in the art and are particularly useful fordelivery of agents at a particular site, such as could be used with theagents of the present invention. In this embodiment, the dosage unitcontains a corresponding multiple of the daily dose.

The effect of a single dose on GNAQ levels is long lasting, such thatsubsequent doses are administered at not more than 3, 4, or 5 dayintervals, or at not more than 1, 2, 3, or 4 week intervals, or at notmore than 5, 6, 7, 8, 9, or 10 week intervals.

The skilled artisan will appreciate that certain factors may influencethe dosage and timing required to effectively treat a subject, includingbut not limited to the severity of the disease or disorder, previoustreatments, the general health and/or age of the subject, and otherdiseases present. Moreover, treatment of a subject with atherapeutically effective amount of a composition can include a singletreatment or a series of treatments. Estimates of effective dosages andin vivo half-lives for the individual dsRNAs encompassed by theinvention can be made using conventional methodologies or on the basisof in vivo testing using an appropriate animal model, as describedelsewhere herein.

Advances in mouse genetics have generated a number of mouse models forthe study of various human diseases, such as pathological processesmediated by GNAQ expression. Such models are used for in vivo testing ofdsRNA, as well as for determining a therapeutically effective dose. Asuitable mouse model is, for example, a mouse containing a plasmidexpressing human GNAQ. Another suitable mouse model is a transgenicmouse carrying a transgene that expresses human GNAQ.

The data obtained from cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofcompositions featured in the invention lies generally within a range ofcirculating concentrations that include the ED50 with little or notoxicity. The dosage may vary within this range depending upon thedosage form employed and the route of administration utilized. For anycompound used in the methods featured in the invention, thetherapeutically effective dose can be estimated initially from cellculture assays. A dose may be formulated in animal models to achieve acirculating plasma concentration range of the compound or, whenappropriate, of the polypeptide product of a target sequence (e.g.,achieving a decreased concentration of the polypeptide) that includesthe IC50 (i.e., the concentration of the test compound which achieves ahalf-maximal inhibition of symptoms) as determined in cell culture. Suchinformation can be used to more accurately determine useful doses inhumans. Levels in plasma may be measured, for example, by highperformance liquid chromatography.

The dsRNAs featured in the invention can be administered in combinationwith other known agents effective in treatment of pathological processesmediated by target gene expression. In any event, the administeringphysician can adjust the amount and timing of dsRNA administration onthe basis of results observed using standard measures of efficacy knownin the art or described herein.

Administration

The present invention also includes pharmaceutical compositions andformulations which include the dsRNA compounds featured in theinvention. The pharmaceutical compositions of the present invention maybe administered in a number of ways depending upon whether local orsystemic treatment is desired and upon the area to be treated.Administration may be topical, pulmonary, e.g., by inhalation orinsufflation of powders or aerosols, including by nebulizer;intratracheal, intranasal, epidermal and transdermal, oral orparenteral. Parenteral administration includes intravenous,intraarterial, subcutaneous, intraperitoneal or intramuscular injectionor infusion; or intracranial, e.g., intraparenchymal, intrathecal orintraventricular, administration.

The dsRNA can be delivered in a manner to target a particular tissue,such as the liver (e.g., the hepatocytes of the liver).

The present invention includes pharmaceutical compositions that can bedelivered by injection directly into the brain. The injection can be bystereotactic injection into a particular region of the brain (e.g., thesubstantia nigra, cortex, hippocampus, striatum, or globus pallidus), orthe dsRNA can be delivered into multiple regions of the central nervoussystem (e.g., into multiple regions of the brain, and/or into the spinalcord). The dsRNA can also be delivered into diffuse regions of the brain(e.g., diffuse delivery to the cortex of the brain).

In one embodiment, a dsRNA targeting GNAQ can be delivered by way of acannula or other delivery device having one end implanted in a tissue,e.g., the brain, e.g., the substantia nigra, cortex, hippocampus,striatum, corpus callosum or globus pallidus of the brain. The cannulacan be connected to a reservoir of the dsRNA composition. The flow ordelivery can be mediated by a pump, e.g., an osmotic pump or minipump,such as an Alzet pump (Durect, Cupertino, Calif.). In one embodiment, apump and reservoir are implanted in an area distant from the tissue,e.g., in the abdomen, and delivery is effected by a conduit leading fromthe pump or reservoir to the site of release. Infusion of the dsRNAcomposition into the brain can be over several hours or for severaldays, e.g., for 1, 2, 3, 5, or 7 days or more. Devices for delivery tothe brain are described, for example, in U.S. Pat. Nos. 6,093,180, and5,814,014.

Pharmaceutical compositions and formulations for topical administrationmay include transdermal patches, ointments, lotions, creams, gels,drops, suppositories, sprays, liquids and powders. Conventionalpharmaceutical carriers, aqueous, powder or oily bases, thickeners andthe like may be necessary or desirable. Coated condoms, gloves and thelike may also be useful. Suitable topical formulations include those inwhich the dsRNAs featured in the invention are in admixture with atopical delivery agent such as lipids, liposomes, fatty acids, fattyacid esters, steroids, chelating agents and surfactants. Suitable lipidsand liposomes include neutral (e.g., dioleoylphosphatidyl DOPEethanolamine, dimyristoylphosphatidyl choline DMPC,distearoylphosphatidyl choline) negative (e.g., dimyristoylphosphatidylglycerol DMPG) and cationic (e.g., dioleoyltetramethylaminopropyl DOTAPand dioleoylphosphatidyl ethanolamine DOTMA). DsRNAs featured in theinvention may be encapsulated within liposomes or may form complexesthereto, in particular to cationic liposomes. Alternatively, dsRNAs maybe complexed to lipids, in particular to cationic lipids. Suitable fattyacids and esters include but are not limited to arachidonic acid, oleicacid, eicosanoic acid, lauric acid, caprylic acid, capric acid, myristicacid, palmitic acid, stearic acid, linoleic acid, linolenic acid,dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate,1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or aC₁₋₁₀ alkyl ester (e.g., isopropylmyristate IPM), monoglyceride,diglyceride or pharmaceutically acceptable salt thereof. Topicalformulations are described in detail in U.S. Pat. No. 6,747,014, whichis incorporated herein by reference.

Liposomal Formulations

There are many organized surfactant structures besides microemulsionsthat have been studied and used for the formulation of drugs. Theseinclude monolayers, micelles, bilayers and vesicles. Vesicles, such asliposomes, have attracted great interest because of their specificityand the duration of action they offer from the standpoint of drugdelivery. As used in the present invention, the term “liposome” means avesicle composed of amphiphilic lipids arranged in a spherical bilayeror bilayers.

Liposomes are unilamellar or multilamellar vesicles which have amembrane formed from a lipophilic material and an aqueous interior. Theaqueous portion contains the composition to be delivered. Cationicliposomes possess the advantage of being able to fuse to the cell wall.Non-cationic liposomes, although not able to fuse as efficiently withthe cell wall, are taken up by macrophages in vivo.

In order to cross intact mammalian skin, lipid vesicles must passthrough a series of fine pores, each with a diameter less than 50 nm,under the influence of a suitable transdermal gradient. Therefore, it isdesirable to use a liposome which is highly deformable and able to passthrough such fine pores.

Further advantages of liposomes include; liposomes obtained from naturalphospholipids are biocompatible and biodegradable; liposomes canincorporate a wide range of water and lipid soluble drugs; liposomes canprotect encapsulated drugs in their internal compartments frommetabolism and degradation (Rosoff, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 245). Important considerations in thepreparation of liposome formulations are the lipid surface charge,vesicle size and the aqueous volume of the liposomes.

Liposomes are useful for the transfer and delivery of active ingredientsto the site of action. Because the liposomal membrane is structurallysimilar to biological membranes, when liposomes are applied to a tissue,the liposomes start to merge with the cellular membranes and as themerging of the liposome and cell progresses, the liposomal contents areemptied into the cell where the active agent may act.

Liposomal formulations have been the focus of extensive investigation asthe mode of delivery for many drugs. There is growing evidence that fortopical administration, liposomes present several advantages over otherformulations. Such advantages include reduced side-effects related tohigh systemic absorption of the administered drug, increasedaccumulation of the administered drug at the desired target, and theability to administer a wide variety of drugs, both hydrophilic andhydrophobic, into the skin.

Several reports have detailed the ability of liposomes to deliver agentsincluding high-molecular weight DNA into the skin. Compounds includinganalgesics, antibodies, hormones and high-molecular weight DNAs havebeen administered to the skin. The majority of applications resulted inthe targeting of the upper epidermis

Liposomes fall into two broad classes. Cationic liposomes are positivelycharged liposomes which interact with the negatively charged DNAmolecules to form a stable complex. The positively charged DNA/liposomecomplex binds to the negatively charged cell surface and is internalizedin an endosome. Due to the acidic pH within the endosome, the liposomesare ruptured, releasing their contents into the cell cytoplasm (Wang etal., Biochem. Biophys. Res. Commun., 1987, 147, 980-985).

Liposomes which are pH-sensitive or negatively-charged, entrap DNArather than complex with it. Since both the DNA and the lipid aresimilarly charged, repulsion rather than complex formation occurs.Nevertheless, some DNA is entrapped within the aqueous interior of theseliposomes. pH-sensitive liposomes have been used to deliver DNA encodingthe thymidine kinase gene to cell monolayers in culture. Expression ofthe exogenous gene was detected in the target cells (Zhou et al.,Journal of Controlled Release, 1992, 19, 269-274).

One major type of liposomal composition includes phospholipids otherthan naturally-derived phosphatidylcholine. Neutral liposomecompositions, for example, can be formed from dimyristoylphosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC).Anionic liposome compositions generally are formed from dimyristoylphosphatidylglycerol, while anionic fusogenic liposomes are formedprimarily from dioleoyl phosphatidylethanolamine (DOPE). Another type ofliposomal composition is formed from phosphatidylcholine (PC) such as,for example, soybean PC, and egg PC. Another type is formed frommixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.

Several studies have assessed the topical delivery of liposomal drugformulations to the skin. Application of liposomes containing interferonto guinea pig skin resulted in a reduction of skin herpes sores whiledelivery of interferon via other means (e.g., as a solution or as anemulsion) were ineffective (Weiner et al., Journal of Drug Targeting,1992, 2, 405-410). Further, an additional study tested the efficacy ofinterferon administered as part of a liposomal formulation to theadministration of interferon using an aqueous system, and concluded thatthe liposomal formulation was superior to aqueous administration (duPlessis et al., Antiviral Research, 1992, 18, 259-265).

Non-ionic liposomal systems have also been examined to determine theirutility in the delivery of drugs to the skin, in particular systemscomprising non-ionic surfactant and cholesterol. Non-ionic liposomalformulations comprising Novasome™ I (glyceryldilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome™ II(glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) wereused to deliver cyclosporin-A into the dermis of mouse skin. Resultsindicated that such non-ionic liposomal systems were effective infacilitating the deposition of cyclosporin-A into different layers ofthe skin (Hu et al. S.T.P.Pharma. Sci., 1994, 4, 6, 466).

Liposomes also include “sterically stabilized” liposomes, a term which,as used herein, refers to liposomes comprising one or more specializedlipids that, when incorporated into liposomes, result in enhancedcirculation lifetimes relative to liposomes lacking such specializedlipids. Examples of sterically stabilized liposomes are those in whichpart of the vesicle-forming lipid portion of the liposome (A) comprisesone or more glycolipids, such as monosialoganglioside G_(M1), or (B) isderivatized with one or more hydrophilic polymers, such as apolyethylene glycol (PEG) moiety. While not wishing to be bound by anyparticular theory, it is thought in the art that, at least forsterically stabilized liposomes containing gangliosides, sphingomyelin,or PEG-derivatized lipids, the enhanced circulation half-life of thesesterically stabilized liposomes derives from a reduced uptake into cellsof the reticuloendothelial system (RES) (Allen et al., FEBS Letters,1987, 223, 42; Wu et al., Cancer Research, 1993, 53, 3765).

Various liposomes comprising one or more glycolipids are known in theart. Papahadjopoulos et al. (Ann. N.Y. Acad. Sci., 1987, 507, 64)reported the ability of monosialoganglioside G_(M1), galactocerebrosidesulfate and phosphatidylinositol to improve blood half-lives ofliposomes. These findings were expounded upon by Gabizon et al. (Proc.Natl. Acad. Sci. U.S.A., 1988, 85, 6949). U.S. Pat. No. 4,837,028 and WO88/04924, both to Allen et al., disclose liposomes comprising (1)sphingomyelin and (2) the ganglioside G_(M1) or a galactocerebrosidesulfate ester. U.S. Pat. No. 5,543,152 (Webb et al.) discloses liposomescomprising sphingomyelin. Liposomes comprising1,2-sn-dimyristoylphosphatidylcholine are disclosed in WO 97/13499 (Limet al).

Many liposomes comprising lipids derivatized with one or morehydrophilic polymers, and methods of preparation thereof, are known inthe art. Sunamoto et al. (Bull. Chem. Soc. Jpn., 1980, 53, 2778)described liposomes comprising a nonionic detergent, 2C_(1215G), thatcontains a PEG moiety. Illum et al. (FEBS Lett., 1984, 167, 79) notedthat hydrophilic coating of polystyrene particles with polymeric glycolsresults in significantly enhanced blood half-lives. Syntheticphospholipids modified by the attachment of carboxylic groups ofpolyalkylene glycols (e.g., PEG) are described by Sears (U.S. Pat. Nos.4,426,330 and 4,534,899). Klibanov et al. (FEBS Lett., 1990, 268, 235)described experiments demonstrating that liposomes comprisingphosphatidylethanolamine (PE) derivatized with PEG or PEG stearate havesignificant increases in blood circulation half-lives. Blume et al.(Biochimica et Biophysica Acta, 1990, 1029, 91) extended suchobservations to other PEG-derivatized phospholipids, e.g., DSPE-PEG,formed from the combination of distearoylphosphatidylethanolamine (DSPE)and PEG. Liposomes having covalently bound PEG moieties on theirexternal surface are described in European Patent No. EP 0 445 131 B1and WO 90/04384 to Fisher. Liposome compositions containing 1-20 molepercent of PE derivatized with PEG, and methods of use thereof, aredescribed by Woodle et al. (U.S. Pat. Nos. 5,013,556 and 5,356,633) andMartin et al. (U.S. Pat. No. 5,213,804 and European Patent No. EP 0 496813 B1). Liposomes comprising a number of other lipid-polymer conjugatesare disclosed in WO 91/05545 and U.S. Pat. No. 5,225,212 (both to Martinet al.) and in WO 94/20073 (Zalipsky et al.) Liposomes comprisingPEG-modified ceramide lipids are described in WO 96/10391 (Choi et al).U.S. Pat. No. 5,540,935 (Miyazaki et al.) and U.S. Pat. No. 5,556,948(Tagawa et al.) describe PEG-containing liposomes that can be furtherderivatized with functional moieties on their surfaces.

A number of liposomes comprising nucleic acids are known in the art. WO96/40062 to Thierry et al. discloses methods for encapsulating highmolecular weight nucleic acids in liposomes. U.S. Pat. No. 5,264,221 toTagawa et al. discloses protein-bonded liposomes and asserts that thecontents of such liposomes may include a dsRNA. U.S. Pat. No. 5,665,710to Rahman et al. describes certain methods of encapsulatingoligodeoxynucleotides in liposomes. WO 97/04787 to Love et al. disclosesliposomes comprising dsRNAs targeted to the raf gene.

Transfersomes are yet another type of liposomes, and are highlydeformable lipid aggregates which are candidates for drug deliveryvehicles. Transfersomes may be described as lipid droplets which are sohighly deformable that they are easily able to penetrate through poreswhich are smaller than the droplet. Transfersomes are adaptable to theenvironment in which they are used, e.g., they are self-optimizing(adaptive to the shape of pores in the skin), self-repairing, frequentlyreach their targets without fragmenting, and often self-loading. To maketransfersomes it is possible to add surface edge-activators, usuallysurfactants, to a standard liposomal composition. Transfersomes havebeen used to deliver serum albumin to the skin. Thetransfersome-mediated delivery of serum albumin has been shown to be aseffective as subcutaneous injection of a solution containing serumalbumin.

Surfactants find wide application in formulations such as emulsions(including microemulsions) and liposomes. The most common way ofclassifying and ranking the properties of the many different types ofsurfactants, both natural and synthetic, is by the use of thehydrophile/lipophile balance (HLB). The nature of the hydrophilic group(also known as the “head”) provides the most useful means forcategorizing the different surfactants used in formulations (Rieger, inPharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988,p. 285).

If the surfactant molecule is not ionized, it is classified as anonionic surfactant. Nonionic surfactants find wide application inpharmaceutical and cosmetic products and are usable over a wide range ofpH values. In general their HLB values range from 2 to about 18depending on their structure. Nonionic surfactants include nonionicesters such as ethylene glycol esters, propylene glycol esters, glycerylesters, polyglyceryl esters, sorbitan esters, sucrose esters, andethoxylated esters. Nonionic alkanolamides and ethers such as fattyalcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylatedblock polymers are also included in this class. The polyoxyethylenesurfactants are the most popular members of the nonionic surfactantclass.

If the surfactant molecule carries a negative charge when it isdissolved or dispersed in water, the surfactant is classified asanionic. Anionic surfactants include carboxylates such as soaps, acyllactylates, acyl amides of amino acids, esters of sulfuric acid such asalkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkylbenzene sulfonates, acyl isethionates, acyl taurates andsulfosuccinates, and phosphates. The most important members of theanionic surfactant class are the alkyl sulfates and the soaps.

If the surfactant molecule carries a positive charge when it isdissolved or dispersed in water, the surfactant is classified ascationic. Cationic surfactants include quaternary ammonium salts andethoxylated amines. The quaternary ammonium salts are the most usedmembers of this class.

If the surfactant molecule has the ability to carry either a positive ornegative charge, the surfactant is classified as amphoteric. Amphotericsurfactants include acrylic acid derivatives, substituted alkylamides,N-alkylbetaines and phosphatides.

The use of surfactants in drug products, formulations and in emulsionshas been reviewed (Rieger, in Pharmaceutical Dosage Forms, MarcelDekker, Inc., New York, N.Y., 1988, p. 285).

Nucleic Acid Lipid Particles

In one embodiment, a GNAQ dsRNA featured in the invention is fullyencapsulated in the lipid formulation, e.g., to form a SPLP, pSPLP,SNALP, or other nucleic acid-lipid particle. As used herein, the term“SNALP” refers to a stable nucleic acid-lipid particle, including SPLP.As used herein, the term “SPLP” refers to a nucleic acid-lipid particlecomprising plasmid DNA encapsulated within a lipid vesicle. SNALPs andSPLPs typically contain a cationic lipid, a non-cationic lipid, and alipid that prevents aggregation of the particle (e.g., a PEG-lipidconjugate). SNALPs and SPLPs are extremely useful for systemicapplications, as they exhibit extended circulation lifetimes followingintravenous (i.v.) injection and accumulate at distal sites (e.g., sitesphysically separated from the administration site). SPLPs include“pSPLP,” which include an encapsulated condensing agent-nucleic acidcomplex as set forth in PCT Publication No. WO 00/03683. The particlesof the present invention typically have a mean diameter of about 50 nmto about 150 nm, more typically about 60 nm to about 130 nm, moretypically about 70 nm to about 110 nm, most typically about 70 nm toabout 90 nm, and are substantially nontoxic. In addition, the nucleicacids when present in the nucleic acid-lipid particles of the presentinvention are resistant in aqueous solution to degradation with anuclease. Nucleic acid-lipid particles and their method of preparationare disclosed in, e.g., U.S. Pat. Nos. 5,976,567; 5,981,501; 6,534,484;6,586,410; 6,815,432; and PCT Publication No. WO 96/40964.

In one embodiment, the lipid to drug ratio (mass/mass ratio) (e.g.,lipid to dsRNA ratio) will be in the range of from about 1:1 to about50:1, from about 1:1 to about 25:1, from about 3:1 to about 15:1, fromabout 4:1 to about 10:1, from about 5:1 to about 9:1, or about 6:1 toabout 9:1. In some embodiments the lipid to dsRNA ratio can be about1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, or 11:1.

The cationic lipid may be, for example, N,N-dioleyl-N,N-dimethylammoniumchloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB),N-(I-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP),N-(I-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA),N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA),1,2-DiLinoleyloxy-N,N-dimethylaminopropane (DLinDMA),1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA),1,2-Dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP),1,2-Dilinoleyoxy-3-(dimethylamino)acetoxypropane (DLin-DAC),1,2-Dilinoleyoxy-3-morpholinopropane (DLin-MA),1,2-Dilinoleoyl-3-dimethylaminopropane (DLinDAP),1,2-Dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA),1-Linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP),1,2-Dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl),1,2-Dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl),1,2-Dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), or3-(N,N-Dilinoleylamino)-1,2-propanediol (DLinAP),3-(N,N-Dioleylamino)-1,2-propanedio (DOAP),1,2-Dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA),1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA),2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA) oranalogs thereof,(3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine(ALN100), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl4-(dimethylamino)butanoate (MC3),1,1′-(2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethylazanediyOdidodecan-2-ol(Tech G1), or a mixture thereof. The cationic lipid may comprise fromabout 20 mol % to about 50 mol % or about 40 mol % of the total lipidpresent in the particle.

The non-cationic lipid may be an anionic lipid or a neutral lipidincluding, but not limited to, distearoylphosphatidylcholine (DSPC),dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine(DPPC), dioleoylphosphatidylglycerol (DOPG),dipalmitoylphosphatidylglycerol (DPPG),dioleoyl-phosphatidylethanolamine (DOPE),palmitoyloleoylphosphatidylcholine (POPC),palmitoyloleoylphosphatidylethanolamine (POPE),dioleoyl-phosphatidylethanolamine4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE),distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethyl PE,16-O-dimethyl PE, 18-1-trans PE,1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), cholesterol, or amixture thereof. The non-cationic lipid may be from about 5 mol % toabout 90 mol %, about 10 mol %, or about 58 mol % if cholesterol isincluded, of the total lipid present in the particle.

The conjugated lipid that inhibits aggregation of particles may be, forexample, a polyethyleneglycol (PEG)-lipid including, without limitation,a PEG-diacylglycerol (DAG), a PEG-dialkyloxypropyl (DAA), aPEG-phospholipid, a PEG-ceramide (Cer), or a mixture thereof. ThePEG-DAA conjugate may be, for example, a PEG-dilauryloxypropyl (Ci₂), aPEG-dimyristyloxypropyl (Ci₄), a PEG-dipalmityloxypropyl (C1₆), or aPEG-distearyloxypropyl (C₁₈). Other examples of PEG conjugates includePEG-cDMA (N-[(methoxy poly(ethyleneglycol)2000)carbamyl]-1,2-dimyristyloxlpropyl-3-amine), mPEG2000-DMG(mPEG-dimyrystylglycerol (with an average molecular weight of 2,000) andPEG-C-DOMG (R-3-[(ω-methoxy-poly (ethyleneglycol)2000)carbamoyl)]-1,2-dimyristyloxlpropyl-3-amine). The conjugatedlipid that prevents aggregation of particles may be from 0 mol % toabout 20 mol % or about 1.0, 1.1., 1.2, 0.13, 1.4, 1.5, 1.6, 1.7, 1.8,1.9, or 2 mol % of the total lipid present in the particle.

In some embodiments, the nucleic acid-lipid particle further includescholesterol at, e.g., about 10 mol % to about 60 mol % or about 48 mol %of the total lipid present in the particle.

In one embodiment, the compound2,2-Dilinoleyl-4-dimethylaminoethyl[1,3]-dioxolane can be used toprepare lipid-siRNA nanoparticles. Synthesis of2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane is described in U.S.provisional patent application No. 61/107,998 filed on Oct. 23, 2008,which is herein incorporated by reference.

For example, the lipid-siRNA particle can include 40% 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane: 10% DSPC: 40%Cholesterol: 10% PEG-C-DOMG (mole percent) with a particle size of63.0±20 nm and a 0.027 siRNA/Lipid Ratio.

In still another embodiment, the compound1,1′-(2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethylazanediyl)didodecan-2-ol(Tech G1) can be used to prepare lipid-siRNA particles. For example, thedsRNA can be formulated in a lipid formulation comprising Tech-G1,distearoyl phosphatidylcholine (DSPC), cholesterol and mPEG2000-DMG at amolar ratio of 50:10:38.5:1.5 ata total lipid to siRNA ratio of 7:1(wt:wt).

LNP01

In one embodiment, the lipidoid ND98.4HCl (MW 1487) (Formula 1),Cholesterol (Sigma-Aldrich), and PEG-Ceramide C16 (Avanti Polar Lipids)can be used to prepare lipid-siRNA nanoparticles (i.e., LNP01particles). Stock solutions of each in ethanol can be prepared asfollows: ND98, 133 mg/ml; Cholesterol, 25 mg/ml, PEG-Ceramide C16, 100mg/ml. The ND98, Cholesterol, and PEG-Ceramide C16 stock solutions canthen be combined in a, e.g., 42:48:10 molar ratio. The combined lipidsolution can be mixed with aqueous siRNA (e.g., in sodium acetate pH 5)such that the final ethanol concentration is about 35-45% and the finalsodium acetate concentration is about 100-300 mM. Lipid-siRNAnanoparticles typically form spontaneously upon mixing. Depending on thedesired particle size distribution, the resultant nanoparticle mixturecan be extruded through a polycarbonate membrane (e.g., 100 nm cut-off)using, for example, a thermobarrel extruder, such as Lipex Extruder(Northern Lipids, Inc). In some cases, the extrusion step can beomitted. Ethanol removal and simultaneous buffer exchange can beaccomplished by, for example, dialysis or tangential flow filtration.Buffer can be exchanged with, for example, phosphate buffered saline(PBS) at about pH 7, e.g., about pH 6.9, about pH 7.0, about pH 7.1,about pH 7.2, about pH 7.3, or about pH 7.4.

LNP01 formulations are described, e.g., in International ApplicationPublication No. WO 2008/042973, which is hereby incorporated byreference.

Additional exemplary lipid-siRNA formulations are as follows:

cationic lipid/non-cationic lipid/ cholesterol/PEG-lipid conjugateCationic Lipid Lipid:siRNA ratio Process SNALP 1,2-Dilinolenyloxy-N,N-DLinDMA/DPPC/Cholesterol/PEG-cDMA dimethylaminopropane(57.1/7.1/34.4/1.4) (DLinDMA) lipid:siRNA ~ 7:1 SNALP-2,2-Dilinoleyl-4-dimethylaminoethyl- XTC/DPPC/Cholesterol/PEG-cDMA XTC[1,3]-dioxolane (XTC) 57.1/7.1/34.4/1.4 lipid:siRNA ~ 7:1 LNP052,2-Dilinoleyl-4-dimethylaminoethyl- XTC/DSPC/Cholesterol/PEG-DMGExtrusion [1,3]-dioxolane (XTC) 57.5/7.5/31.5/3.5 lipid:siRNA ~ 6:1LNP06 2,2-Dilinoleyl-4-dimethylaminoethyl- XTC/DSPC/Cholesterol/PEG-DMGExtrusion [1,3]-dioxolane (XTC) 57.5/7.5/31.5/3.5 lipid:siRNA ~ 11:1LNP07 2,2-Dilinoleyl-4-dimethylaminoethyl- XTC/DSPC/Cholesterol/PEG-DMGIn-line [1,3]-dioxolane (XTC) 60/7.5/31/1.5, mixing lipid:siRNA ~ 6:1LNP08 2,2-Dilinoleyl-4-dimethylaminoethyl- XTC/DSPC/Cholesterol/PEG-DMGIn-line [1,3]-dioxolane (XTC) 60/7.5/31/1.5, mixing lipid:siRNA ~ 11:1LNP09 2,2-Dilinoleyl-4-dimethylaminoethyl- XTC/DSPC/Cholesterol/PEG-DMGIn-line [1,3]-dioxolane (XTC) 50/10/38.5/1.5 mixing Lipid:siRNA 10:1LNP10 (3aR,5s,6aS)-N,N-dimethyl-2,2- ALN100/DSPC/Cholesterol/PEG-DMGIn-line di((9Z,12Z)-octadeca-9,12- 50/10/38.5/1.5 mixingdienyl)tetrahydro-3aH- Lipid:siRNA 10:1 cyclopenta[d][1,3]dioxol-5-amine(ALN100) LNP11 (6Z,9Z,28Z,31Z)-heptatriaconta-MC-3/DSPC/Cholesterol/PEG-DMG In-line 6,9,28,31-tetraen-19-yl 4-50/10/38.5/1.5 mixing (dimethylamino)butanoate (MC3) Lipid:siRNA 10:1LNP12 1,1′-(2-(4-(2-((2-(bis(2- Tech G1/DSPC/Cholesterol/PEG-DMG In-linehydroxydodecyl)amino)ethyl)(2- 50/10/38.5/1.5 mixinghydroxydodecyl)amino)ethyl)piperazin- Lipid:siRNA 10:11-yl)ethylazanediyl)didodecan-2-ol (Tech G1)

LNP09 formulations and XTC comprising formulations are described, e.g.,in U.S. Provisional Ser. No. 61/239,686, filed Sep. 3, 2009, which ishereby incorporated by reference.

LNP11 formulations and MC3 comprising formulations are described, e.g.,in U.S. Provisional Ser. No. 61/244,834, filed Sep. 22, 2009, which ishereby incorporated by reference.

LNP12 formulations and TechG1 comprising formulations are described,e.g., in U.S. Provisional Ser. No. 61/175,770, filed May 5, 2009, whichis hereby incorporated by reference.

Formulations prepared by either the standard or extrusion-free methodcan be characterized in similar manners. For example, formulations aretypically characterized by visual inspection. They should be whitishtranslucent solutions free from aggregates or sediment. Particle sizeand particle size distribution of lipid-nanoparticles can be measured bylight scattering using, for example, a Malvern Zetasizer Nano ZS(Malvern, USA). Particles should be about 20-300 nm, such as 40-100 nmin size. The particle size distribution should be unimodal. The totalsiRNA concentration in the formulation, as well as the entrappedfraction, is estimated using a dye exclusion assay. A sample of theformulated siRNA can be incubated with an RNA-binding dye, such asRibogreen (Molecular Probes) in the presence or absence of a formulationdisrupting surfactant, e.g., 0.5% Triton-X100. The total siRNA in theformulation can be determined by the signal from the sample containingthe surfactant, relative to a standard curve. The entrapped fraction isdetermined by subtracting the “free” siRNA content (as measured by thesignal in the absence of surfactant) from the total siRNA content.Percent entrapped siRNA is typically >85%. For SNALP formulation, theparticle size is at least 30 nm, at least 40 nm, at least 50 nm, atleast 60 nm, at least 70 nm, at least 80 nm, at least 90 nm, at least100 nm, at least 110 nm, and at least 120 nm. The suitable range istypically about at least 50 nm to about at least 110 nm, about at least60 nm to about at least 100 nm, or about at least 80 nm to about atleast 90 nm.

Compositions and formulations for oral administration include powders orgranules, microparticulates, nanoparticulates, suspensions or solutionsin water or non-aqueous media, capsules, gel capsules, sachets, tabletsor minitablets. Thickeners, flavoring agents, diluents, emulsifiers,dispersing aids or binders may be desirable. In some embodiments, oralformulations are those in which dsRNAs featured in the invention areadministered in conjunction with one or more penetration enhancerssurfactants and chelators. Suitable surfactants include fatty acidsand/or esters or salts thereof, bile acids and/or salts thereof.Suitable bile acids/salts include chenodeoxycholic acid (CDCA) andursodeoxychenodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid,deoxycholic acid, glucholic acid, glycholic acid, glycodeoxycholic acid,taurocholic acid, taurodeoxycholic acid, sodiumtauro-24,25-dihydro-fusidate and sodium glycodihydrofusidate. Suitablefatty acids include arachidonic acid, undecanoic acid, oleic acid,lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid,stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate,monoolein, dilaurin, glyceryl 1-monocaprate,1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or amonoglyceride, a diglyceride or a pharmaceutically acceptable saltthereof (e.g., sodium). In some embodiments, combinations of penetrationenhancers are used, for example, fatty acids/salts in combination withbile acids/salts. One exemplary combination is the sodium salt of lauricacid, capric acid and UDCA. Further penetration enhancers includepolyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether. DsRNAsfeatured in the invention may be delivered orally, in granular formincluding sprayed dried particles, or complexed to form micro ornanoparticles. DsRNA complexing agents include poly-amino acids;polyimines; polyacrylates; polyalkylacrylates, polyoxethanes,polyalkylcyanoacrylates; cationized gelatins, albumins, starches,acrylates, polyethyleneglycols (PEG) and starches;polyalkylcyanoacrylates; DEAE-derivatized polyimines, pollulans,celluloses and starches. Suitable complexing agents include chitosan,N-trimethylchitosan, poly-L-lysine, polyhistidine, polyornithine,polyspermines, protamine, polyvinylpyridine,polythiodiethylaminomethylethylene P(TDAE), polyaminostyrene (e.g.,p-amino), poly(methylcyanoacrylate), poly(ethylcyanoacrylate),poly(butylcyanoacrylate), poly(isobutylcyanoacrylate),poly(isohexylcynaoacrylate), DEAE-methacrylate, DEAE-hexylacrylate,DEAE-acrylamide, DEAE-albumin and DEAE-dextran, polymethylacrylate,polyhexylacrylate, poly(D,L-lactic acid), poly(DL-lactic-co-glycolicacid (PLGA), alginate, and polyethyleneglycol (PEG). Oral formulationsfor dsRNAs and their preparation are described in detail in U.S. Pat.No. 6,887,906, US Publn. No. 20030027780, and U.S. Pat. No. 6,747,014,each of which is incorporated herein by reference.

Compositions and formulations for parenteral, intraparenchymal (into thebrain), intrathecal, intraventricular or intrahepatic administration mayinclude sterile aqueous solutions which may also contain buffers,diluents and other suitable additives such as, but not limited to,penetration enhancers, carrier compounds and other pharmaceuticallyacceptable carriers or excipients.

Pharmaceutical compositions of the present invention include, but arenot limited to, solutions, emulsions, and liposome-containingformulations. These compositions may be generated from a variety ofcomponents that include, but are not limited to, preformed liquids,self-emulsifying solids and self-emulsifying semisolids. Particularlypreferred are formulations that target the liver when treating hepaticdisorders such as hepatic carcinoma.

The pharmaceutical formulations of the present invention, which mayconveniently be presented in unit dosage form, may be prepared accordingto conventional techniques well known in the pharmaceutical industry.Such techniques include the step of bringing into association the activeingredients with the pharmaceutical carrier(s) or excipient(s). Ingeneral, the formulations are prepared by uniformly and intimatelybringing into association the active ingredients with liquid carriers orfinely divided solid carriers or both, and then, if necessary, shapingthe product.

The compositions of the present invention may be formulated into any ofmany possible dosage forms such as, but not limited to, tablets,capsules, gel capsules, liquid syrups, soft gels, suppositories, andenemas. The compositions of the present invention may also be formulatedas suspensions in aqueous, non-aqueous or mixed media. Aqueoussuspensions may further contain substances which increase the viscosityof the suspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension may also contain stabilizers.

Emulsions

The compositions of the present invention may be prepared and formulatedas emulsions. Emulsions are typically heterogeneous systems of oneliquid dispersed in another in the form of droplets usually exceeding0.1 μm in diameter (Idson, in Pharmaceutical Dosage Forms, Lieberman,Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y.,volume 1, p. 199; Rosoff, in Pharmaceutical Dosage Forms, Lieberman,Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y.,Volume 1, p. 245; Block in Pharmaceutical Dosage Forms, Lieberman,Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y.,volume 2, p. 335; Higuchi et al., in Remington's PharmaceuticalSciences, Mack Publishing Co., Easton, Pa., 1985, p. 301). Emulsions areoften biphasic systems comprising two immiscible liquid phasesintimately mixed and dispersed with each other. In general, emulsionsmay be of either the water-in-oil (w/o) or the oil-in-water (o/w)variety. When an aqueous phase is finely divided into and dispersed asminute droplets into a bulk oily phase, the resulting composition iscalled a water-in-oil (w/o) emulsion. Alternatively, when an oily phaseis finely divided into and dispersed as minute droplets into a bulkaqueous phase, the resulting composition is called an oil-in-water (o/w)emulsion. Emulsions may contain additional components in addition to thedispersed phases, and the active drug which may be present as a solutionin either the aqueous phase, oily phase or itself as a separate phase.Pharmaceutical excipients such as emulsifiers, stabilizers, dyes, andanti-oxidants may also be present in emulsions as needed. Pharmaceuticalemulsions may also be multiple emulsions that are comprised of more thantwo phases such as, for example, in the case of oil-in-water-in-oil(o/w/o) and water-in-oil-in-water (w/o/w) emulsions. Such complexformulations often provide certain advantages that simple binaryemulsions do not. Multiple emulsions in which individual oil droplets ofan o/w emulsion enclose small water droplets constitute a w/o/wemulsion. Likewise a system of oil droplets enclosed in globules ofwater stabilized in an oily continuous phase provides an o/w/o emulsion.

Emulsions are characterized by little or no thermodynamic stability.Often, the dispersed or discontinuous phase of the emulsion is welldispersed into the external or continuous phase and maintained in thisform through the means of emulsifiers or the viscosity of theformulation. Either of the phases of the emulsion may be a semisolid ora solid, as is the case of emulsion-style ointment bases and creams.Other means of stabilizing emulsions entail the use of emulsifiers thatmay be incorporated into either phase of the emulsion. Emulsifiers maybroadly be classified into four categories: synthetic surfactants,naturally occurring emulsifiers, absorption bases, and finely dispersedsolids (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger andBanker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p.199).

Synthetic surfactants, also known as surface active agents, have foundwide applicability in the formulation of emulsions and have beenreviewed in the literature (Rieger, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 285; Idson, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), Marcel Dekker, Inc., New York,N.Y., 1988, volume 1, p. 199). Surfactants are typically amphiphilic andcomprise a hydrophilic and a hydrophobic portion. The ratio of thehydrophilic to the hydrophobic nature of the surfactant has been termedthe hydrophile/lipophile balance (HLB) and is a valuable tool incategorizing and selecting surfactants in the preparation offormulations. Surfactants may be classified into different classes basedon the nature of the hydrophilic group: nonionic, anionic, cationic andamphoteric (Rieger, in Pharmaceutical Dosage Forms, Lieberman, Riegerand Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1,p. 285).

Naturally occurring emulsifiers used in emulsion formulations includelanolin, beeswax, phosphatides, lecithin and acacia. Absorption basespossess hydrophilic properties such that they can soak up water to formw/o emulsions yet retain their semisolid consistencies, such asanhydrous lanolin and hydrophilic petrolatum. Finely divided solids havealso been used as good emulsifiers especially in combination withsurfactants and in viscous preparations. These include polar inorganicsolids, such as heavy metal hydroxides, nonswelling clays such asbentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidalaluminum silicate and colloidal magnesium aluminum silicate, pigmentsand nonpolar solids such as carbon or glyceryl tristearate.

A large variety of non-emulsifying materials are also included inemulsion formulations and contribute to the properties of emulsions.These include fats, oils, waxes, fatty acids, fatty alcohols, fattyesters, humectants, hydrophilic colloids, preservatives and antioxidants(Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335;Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

Hydrophilic colloids or hydrocolloids include naturally occurring gumsand synthetic polymers such as polysaccharides (for example, acacia,agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth),cellulose derivatives (for example, carboxymethylcellulose andcarboxypropylcellulose), and synthetic polymers (for example, carbomers,cellulose ethers, and carboxyvinyl polymers). These disperse or swell inwater to form colloidal solutions that stabilize emulsions by formingstrong interfacial films around the dispersed-phase droplets and byincreasing the viscosity of the external phase.

Since emulsions often contain a number of ingredients such ascarbohydrates, proteins, sterols and phosphatides that may readilysupport the growth of microbes, these formulations often incorporatepreservatives. Commonly used preservatives included in emulsionformulations include methyl paraben, propyl paraben, quaternary ammoniumsalts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boricacid. Antioxidants are also commonly added to emulsion formulations toprevent deterioration of the formulation. Antioxidants used may be freeradical scavengers such as tocopherols, alkyl gallates, butylatedhydroxyanisole, butylated hydroxytoluene, or reducing agents such asascorbic acid and sodium metabisulfite, and antioxidant synergists suchas citric acid, tartaric acid, and lecithin.

The application of emulsion formulations via dermatological, oral andparenteral routes and methods for their manufacture have been reviewedin the literature (Idson, in Pharmaceutical Dosage Forms, Lieberman,Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y.,volume 1, p. 199). Emulsion formulations for oral delivery have beenvery widely used because of ease of formulation, as well as efficacyfrom an absorption and bioavailability standpoint (Rosoff, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Idson, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Mineral-oil baselaxatives, oil-soluble vitamins and high fat nutritive preparations areamong the materials that have commonly been administered orally as o/wemulsions.

In one embodiment of the present invention, the compositions of dsRNAsand nucleic acids are formulated as microemulsions. A microemulsion maybe defined as a system of water, oil and amphiphile which is a singleoptically isotropic and thermodynamically stable liquid solution(Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245).Typically microemulsions are systems that are prepared by firstdispersing an oil in an aqueous surfactant solution and then adding asufficient amount of a fourth component, generally an intermediatechain-length alcohol to form a transparent system. Therefore,microemulsions have also been described as thermodynamically stable,isotropically clear dispersions of two immiscible liquids that arestabilized by interfacial films of surface-active molecules (Leung andShah, in: Controlled Release of Drugs: Polymers and Aggregate Systems,Rosoff, M., Ed., 1989, VCH Publishers, New York, pages 185-215).Microemulsions commonly are prepared via a combination of three to fivecomponents that include oil, water, surfactant, cosurfactant andelectrolyte. Whether the microemulsion is of the water-in-oil (w/o) oran oil-in-water (o/w) type is dependent on the properties of the oil andsurfactant used and on the structure and geometric packing of the polarheads and hydrocarbon tails of the surfactant molecules (Schott, inRemington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa.,1985, p. 271).

The phenomenological approach utilizing phase diagrams has beenextensively studied and has yielded a comprehensive knowledge, to oneskilled in the art, of how to formulate microemulsions (Rosoff, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Block, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335). Compared toconventional emulsions, microemulsions offer the advantage ofsolubilizing water-insoluble drugs in a formulation of thermodynamicallystable droplets that are formed spontaneously.

Surfactants used in the preparation of microemulsions include, but arenot limited to, ionic surfactants, non-ionic surfactants, Brij 96,polyoxyethylene oleyl ethers, polyglycerol fatty acid esters,tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310),hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500),decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750),decaglycerol sequioleate (SO750), decaglycerol decaoleate (DAO750),alone or in combination with cosurfactants. The cosurfactant, usually ashort-chain alcohol such as ethanol, 1-propanol, and 1-butanol, servesto increase the interfacial fluidity by penetrating into the surfactantfilm and consequently creating a disordered film because of the voidspace generated among surfactant molecules. Microemulsions may, however,be prepared without the use of cosurfactants and alcohol-freeself-emulsifying microemulsion systems are known in the art. The aqueousphase may typically be, but is not limited to, water, an aqueoussolution of the drug, glycerol, PEG300, PEG400, polyglycerols, propyleneglycols, and derivatives of ethylene glycol. The oil phase may include,but is not limited to, materials such as Captex 300, Captex 355, CapmulMCM, fatty acid esters, medium chain (C8-C12) mono, di, andtri-glycerides, polyoxyethylated glyceryl fatty acid esters, fattyalcohols, polyglycolized glycerides, saturated polyglycolized C8-C10glycerides, vegetable oils and silicone oil.

Microemulsions are particularly of interest from the standpoint of drugsolubilization and the enhanced absorption of drugs. Lipid basedmicroemulsions (both o/w and w/o) have been proposed to enhance the oralbioavailability of drugs, including peptides (Constantinides et al.,Pharmaceutical Research, 1994, 11, 1385-1390; Ritschel, Meth. Find. Exp.Clin. Pharmacol., 1993, 13, 205). Microemulsions afford advantages ofimproved drug solubilization, protection of drug from enzymatichydrolysis, possible enhancement of drug absorption due tosurfactant-induced alterations in membrane fluidity and permeability,ease of preparation, ease of oral administration over solid dosageforms, improved clinical potency, and decreased toxicity (Constantinideset al., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J. Pharm.Sci., 1996, 85, 138-143). Often microemulsions may form spontaneouslywhen their components are brought together at ambient temperature. Thismay be particularly advantageous when formulating thermolabile drugs,peptides or dsRNAs. Microemulsions have also been effective in thetransdermal delivery of active components in both cosmetic andpharmaceutical applications. It is expected that the microemulsioncompositions and formulations of the present invention will facilitatethe increased systemic absorption of dsRNAs and nucleic acids from thegastrointestinal tract, as well as improve the local cellular uptake ofdsRNAs and nucleic acids.

Microemulsions of the present invention may also contain additionalcomponents and additives such as sorbitan monostearate (Grill 3),Labrasol, and penetration enhancers to improve the properties of theformulation and to enhance the absorption of the dsRNAs and nucleicacids of the present invention. Penetration enhancers used in themicroemulsions of the present invention may be classified as belongingto one of five broad categories—surfactants, fatty acids, bile salts,chelating agents, and non-chelating non-surfactants (Lee et al.,Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Eachof these classes has been discussed above.

Penetration Enhancers

In one embodiment, the present invention employs various penetrationenhancers to effect the efficient delivery of nucleic acids,particularly dsRNAs, to the skin of animals. Most drugs are present insolution in both ionized and nonionized forms. However, usually onlylipid soluble or lipophilic drugs readily cross cell membranes. It hasbeen discovered that even non-lipophilic drugs may cross cell membranesif the membrane to be crossed is treated with a penetration enhancer. Inaddition to aiding the diffusion of non-lipophilic drugs across cellmembranes, penetration enhancers also enhance the permeability oflipophilic drugs.

Penetration enhancers may be classified as belonging to one of fivebroad categories, i.e., surfactants, fatty acids, bile salts, chelatingagents, and non-chelating non-surfactants (Lee et al., Critical Reviewsin Therapeutic Drug Carrier Systems, 1991, p. 92). Each of the abovementioned classes of penetration enhancers are described below ingreater detail.

Surfactants: In connection with the present invention, surfactants (or“surface-active agents”) are chemical entities which, when dissolved inan aqueous solution, reduce the surface tension of the solution or theinterfacial tension between the aqueous solution and another liquid,with the result that absorption of dsRNAs through the mucosa isenhanced. In addition to bile salts and fatty acids, these penetrationenhancers include, for example, sodium lauryl sulfate,polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether) (Leeet al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92); and perfluorochemical emulsions, such as FC-43. Takahashi et al.,J. Pharm. Pharmacol., 1988, 40, 252).

Fatty acids: Various fatty acids and their derivatives which act aspenetration enhancers include, for example, oleic acid, lauric acid,capric acid (n-decanoic acid), myristic acid, palmitic acid, stearicacid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein(1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid,glycerol 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines,acylcholines, C.sub.1-10 alkyl esters thereof (e.g., methyl, isopropyland t-butyl), and mono- and di-glycerides thereof (i.e., oleate,laurate, caprate, myristate, palmitate, stearate, linoleate, etc.) (Leeet al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems,1990, 7, 1-33; El Hariri et al., J. Pharm. Pharmacol., 1992, 44,651-654).

Bile salts: The physiological role of bile includes the facilitation ofdispersion and absorption of lipids and fat-soluble vitamins (Brunton,Chapter 38 in: Goodman & Gilman's The Pharmacological Basis ofTherapeutics, 9th Ed., Hardman et al. Eds., McGraw-Hill, New York, 1996,pp. 934-935). Various natural bile salts, and their syntheticderivatives, act as penetration enhancers. Thus the term “bile salts”includes any of the naturally occurring components of bile as well asany of their synthetic derivatives. Suitable bile salts include, forexample, cholic acid (or its pharmaceutically acceptable sodium salt,sodium cholate), dehydrocholic acid (sodium dehydrocholate), deoxycholicacid (sodium deoxycholate), glucholic acid (sodium glucholate),glycholic acid (sodium glycocholate), glycodeoxycholic acid (sodiumglycodeoxycholate), taurocholic acid (sodium taurocholate),taurodeoxycholic acid (sodium taurodeoxycholate), chenodeoxycholic acid(sodium chenodeoxycholate), ursodeoxycholic acid (UDCA), sodiumtauro-24,25-dihydro-fusidate (STDHF), sodium glycodihydrofusidate andpolyoxyethylene-9-lauryl ether (POE) (Lee et al., Critical Reviews inTherapeutic Drug Carrier Systems, 1991, page 92; Swinyard, Chapter 39In: Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., MackPublishing Co., Easton, Pa., 1990, pages 782-783; Muranishi, CriticalReviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Yamamoto etal., J. Pharm. Exp. Ther., 1992, 263, 25; Yamashita et al., J. Pharm.Sci., 1990, 79, 579-583).

Chelating Agents: Chelating agents, as used in connection with thepresent invention, can be defined as compounds that remove metallic ionsfrom solution by forming complexes therewith, with the result thatabsorption of dsRNAs through the mucosa is enhanced. With regards totheir use as penetration enhancers in the present invention, chelatingagents have the added advantage of also serving as DNase inhibitors, asmost characterized DNA nucleases require a divalent metal ion forcatalysis and are thus inhibited by chelating agents (Jarrett, J.Chromatogr., 1993, 618, 315-339). Suitable chelating agents include butare not limited to disodium ethylenediaminetetraacetate (EDTA), citricacid, salicylates (e.g., sodium salicylate, 5-methoxysalicylate andhomovanilate), N-acyl derivatives of collagen, laureth-9 and N-aminoacyl derivatives of beta-diketones (enamines)(Lee et al., CriticalReviews in Therapeutic Drug Carrier Systems, 1991, page 92; Muranishi,Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33;Buur et al., J. Control Rel., 1990, 14, 43-51).

Non-chelating non-surfactants: As used herein, non-chelatingnon-surfactant penetration enhancing compounds can be defined ascompounds that demonstrate insignificant activity as chelating agents oras surfactants but that nonetheless enhance absorption of dsRNAs throughthe alimentary mucosa (Muranishi, Critical Reviews in Therapeutic DrugCarrier Systems, 1990, 7, 1-33). This class of penetration enhancersinclude, for example, unsaturated cyclic ureas, 1-alkyl- and1-alkenylazacyclo-alkanone derivatives (Lee et al., Critical Reviews inTherapeutic Drug Carrier Systems, 1991, page 92); and non-steroidalanti-inflammatory agents such as diclofenac sodium, indomethacin andphenylbutazone (Yamashita et al., J. Pharm. Pharmacol., 1987, 39,621-626).

Carriers

Certain compositions of the present invention also incorporate carriercompounds in the formulation. As used herein, “carrier compound” or“carrier” can refer to a nucleic acid, or analog thereof, which is inert(i.e., does not possess biological activity per se) but is recognized asa nucleic acid by in vivo processes that reduce the bioavailability of anucleic acid having biological activity by, for example, degrading thebiologically active nucleic acid or promoting its removal fromcirculation. The coadministration of a nucleic acid and a carriercompound, typically with an excess of the latter substance, can resultin a substantial reduction of the amount of nucleic acid recovered inthe liver, kidney or other extracirculatory reservoirs, presumably dueto competition between the carrier compound and the nucleic acid for acommon receptor. For example, the recovery of a partiallyphosphorothioate dsRNA in hepatic tissue can be reduced when it iscoadministered with polyinosinic acid, dextran sulfate, polycytidic acidor 4-acetamido-4′isothiocyano-stilbene-2,2′-disulfonic acid (Miyao etal., DsRNA Res. Dev., 1995, 5, 115-121; Takakura et al., DsRNA & Nucl.Acid Drug Dev., 1996, 6, 177-183.

Excipients

In contrast to a carrier compound, a “pharmaceutical carrier” or“excipient” is a pharmaceutically acceptable solvent, suspending agentor any other pharmacologically inert vehicle for delivering one or morenucleic acids to an animal. The excipient may be liquid or solid and isselected, with the planned manner of administration in mind, so as toprovide for the desired bulk, consistency, etc., when combined with anucleic acid and the other components of a given pharmaceuticalcomposition. Typical pharmaceutical carriers include, but are notlimited to, binding agents (e.g., pregelatinized maize starch,polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers(e.g., lactose and other sugars, microcrystalline cellulose, pectin,gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calciumhydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc,silica, colloidal silicon dioxide, stearic acid, metallic stearates,hydrogenated vegetable oils, corn starch, polyethylene glycols, sodiumbenzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodiumstarch glycolate, etc.); and wetting agents (e.g., sodium laurylsulphate, etc).

Pharmaceutically acceptable organic or inorganic excipients suitable fornon-parenteral administration which do not deleteriously react withnucleic acids can also be used to formulate the compositions of thepresent invention. Suitable pharmaceutically acceptable carriersinclude, but are not limited to, water, salt solutions, alcohols,polyethylene glycols, gelatin, lactose, amylose, magnesium stearate,talc, silicic acid, viscous paraffin, hydroxymethylcellulose,polyvinylpyrrolidone and the like.

Formulations for topical administration of nucleic acids may includesterile and non-sterile aqueous solutions, non-aqueous solutions incommon solvents such as alcohols, or solutions of the nucleic acids inliquid or solid oil bases. The solutions may also contain buffers,diluents and other suitable additives. Pharmaceutically acceptableorganic or inorganic excipients suitable for non-parenteraladministration which do not deleteriously react with nucleic acids canbe used.

Suitable pharmaceutically acceptable excipients include, but are notlimited to, water, salt solutions, alcohol, polyethylene glycols,gelatin, lactose, amylose, magnesium stearate, talc, silicic acid,viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and thelike.

Other Components

The compositions of the present invention may additionally contain otheradjunct components conventionally found in pharmaceutical compositions,at their art-established usage levels. Thus, for example, thecompositions may contain additional, compatible, pharmaceutically-activematerials such as, for example, antipruritics, astringents, localanesthetics or anti-inflammatory agents, or may contain additionalmaterials useful in physically formulating various dosage forms of thecompositions of the present invention, such as dyes, flavoring agents,preservatives, antioxidants, opacifiers, thickening agents andstabilizers. However, such materials, when added, should not undulyinterfere with the biological activities of the components of thecompositions of the present invention. The formulations can besterilized and, if desired, mixed with auxiliary agents, e.g.,lubricants, preservatives, stabilizers, wetting agents, emulsifiers,salts for influencing osmotic pressure, buffers, colorings, flavoringsand/or aromatic substances and the like which do not deleteriouslyinteract with the nucleic acid(s) of the formulation.

Aqueous suspensions may contain substances which increase the viscosityof the suspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension may also contain stabilizers.

In some embodiments, pharmaceutical compositions featured in theinvention include (a) one or more dsRNA compounds and (b) one or moreanti-cytokine biologic agents which function by a non-RNAi mechanism.Examples of such biologics include, biologics that target IL1β (e.g.,anakinra), IL6 (tocilizumab), or TNF (etanercept, infliximab, adlimumab,or certolizumab).

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD50 (the dose lethal to 50% of thepopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD50/ED50.Compounds that exhibit high therapeutic indices are preferred.

The data obtained from cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofcompositions featured in the invention lies generally within a range ofcirculating concentrations that include the ED50 with little or notoxicity. The dosage may vary within this range depending upon thedosage form employed and the route of administration utilized. For anycompound used in the methods featured in the invention, thetherapeutically effective dose can be estimated initially from cellculture assays. A dose may be formulated in animal models to achieve acirculating plasma concentration range of the compound or, whenappropriate, of the polypeptide product of a target sequence (e.g.,achieving a decreased concentration of the polypeptide) that includesthe IC50 (i.e., the concentration of the test compound which achieves ahalf-maximal inhibition of symptoms) as determined in cell culture. Suchinformation can be used to more accurately determine useful doses inhumans. Levels in plasma may be measured, for example, by highperformance liquid chromatography.

In addition to their administration, as discussed above, the dsRNAsfeatured in the invention can be administered in combination with otherknown agents effective in treatment of pathological processes mediatedby GNAQ expression. In any event, the administering physician can adjustthe amount and timing of dsRNA administration on the basis of resultsobserved using standard measures of efficacy known in the art ordescribed herein.

Methods for Treating Diseases Caused by Expression of a GNAQ Gene

The invention relates in particular to the use of a dsRNA targeting GNAQand compositions containing at least one such dsRNA for the treatment ofa GNAQ-mediated disorder or disease. For example, a dsRNA targeting aGNAQ gene can be useful for the treatment of cancers that have either anactivating mutation of GNAQ and/or are the result of overexpression ofGNAQ. Tumors to be targeted include uveal melanoma, cutaneous melanoma,Blue nevi, Nevi of Ota, and neuroendocrine tumors (including but notlimited to carcinoid tumors, large cell lung cancer, and small cell lungcancer).

A dsRNA targeting a GNAQ gene is also used for treatment of symptoms ofdisorders, such as uveal melanoma. Symptoms associated include, e.g.,melanoma progression, increasing eye pressure, pain in the eye, andimpaired peripheral vision.

Owing to the inhibitory effects on GNAQ expression, a compositionaccording to the invention or a pharmaceutical composition preparedtherefrom can enhance the quality of life.

The invention further relates to the use of a dsRNA or a pharmaceuticalcomposition thereof, e.g., for treating a GNAQ mediated disorder ordisease, in combination with other pharmaceuticals and/or othertherapeutic methods, e.g., with known pharmaceuticals and/or knowntherapeutic methods, such as, for example, those which are currentlyemployed for treating these disorders. In one example, a dsRNA targetingGNAQ can be administered in combination with radiation therapy. In otherexamples, a dsRNA targeting GNAQ can be administered in combination witha pharmaceutical or therapeutic method for treating a symptom of a GNAQdisease, such as pain medication.

The dsRNA and an additional therapeutic agent can be administered in thesame combination, e.g., parenterally, or the additional therapeuticagent can be administered as part of a separate composition or byanother method described herein.

The invention features a method of administering a dsRNA targeting GNAQto a patient having a disease or disorder mediated by GNAQ expression,such as a uveal melanoma. Administration of the dsRNA can stabilize andimprove vision, for example, in a patient with uveal melanoma. Patientscan be administered a therapeutic amount of dsRNA, such as 0.5 mg/kg,1.0 mg/kg, 1.5 mg/kg, 2.0 mg/kg, or 2.5 mg/kg dsRNA. The dsRNA can beadministered by intravenous infusion over a period of time, such as overa 5 minute, 10 minute, 15 minute, 20 minute, or 25 minute period. Theadministration is repeated, for example, on a regular basis, such asbiweekly (i.e., every two weeks) for one month, two months, threemonths, four months or longer. After an initial treatment regimen, thetreatments can be administered on a less frequent basis. For example,after administration biweekly for three months, administration can berepeated once per month, for six months or a year or longer.Administration of the dsRNA can reduce GNAQ levels in the blood or urineof the patient by at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80% or 90%or more.

Before administration of a full dose of the dsRNA, patients can beadministered a smaller dose, such as a 5% infusion reaction, andmonitored for adverse effects, such as an allergic reaction.

Many GNAQ-associated diseases and disorders are hereditary. Therefore, apatient in need of a GNAQ dsRNA can be identified by taking a familyhistory. A healthcare provider, such as a doctor, nurse, or familymember, can take a family history before prescribing or administering aGNAQ dsRNA. A DNA test may also be performed on the patient to identifya mutation in the GNAQ gene, before a GNAQ dsRNA is administered to thepatient.

Methods for Inhibiting Expression of a GNAQ Gene

In yet another aspect, the invention provides a method for inhibitingthe expression of a GNAQ gene in a mammal. The method includesadministering a composition featured in the invention to the mammal suchthat expression of the target GNAQ gene is reduced or silenced.

When the organism to be treated is a mammal such as a human, thecomposition may be administered by any means known in the art including,but not limited to oral or parenteral routes, including intracranial(e.g., intraventricular, intraparenchymal and intrathecal), intravenous,intramuscular, subcutaneous, transdermal, airway (aerosol), nasal,rectal, and topical (including buccal and sublingual) administration. Incertain embodiments, the compositions are administered by intravenousinfusion or injection.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the dsRNAs and methods featured in the invention,suitable methods and materials are described below. All publications,patent applications, patents, and other references mentioned herein areincorporated by reference in their entirety. In case of conflict, thepresent specification, including definitions, will control. In addition,the materials, methods, and examples are illustrative only and notintended to be limiting.

EXAMPLES

Below are examples of specific embodiments for carrying out the presentinvention. The examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.Efforts have been made to ensure accuracy with respect to numbers used(e.g., amounts, temperatures, etc.), but some experimental error anddeviation should, of course, be allowed for.

Other embodiments are, for example, in the claims.

The practice of the present invention will employ, unless otherwiseindicated, conventional methods of protein chemistry, biochemistry,recombinant DNA techniques and pharmacology, within the skill of theart. Such techniques are explained fully in the literature. See, e.g.,T. E. Creighton, Proteins: Structures and Molecular Properties (W.H.Freeman and Company, 1993); A. L. Lehninger, Biochemistry (WorthPublishers, Inc., current addition); Sambrook, et al., MolecularCloning: A Laboratory Manual (2nd Edition, 1989); Methods In Enzymology(S. Colowick and N. Kaplan eds., Academic Press, Inc.); Remington'sPharmaceutical Sciences, 18th Edition (Easton, Pa.: Mack PublishingCompany, 1990); Carey and Sundberg Advanced Organic Chemistry 3rd Ed.(Plenum Press) Vols A and B(1992).

Example 1 dsRNA Synthesis

Source of Reagents

Where the source of a reagent is not specifically given herein, suchreagent may be obtained from any supplier of reagents for molecularbiology at a quality/purity standard for application in molecularbiology.

Conjugates

For the synthesis of 3′-cholesterol-conjugated siRNAs (herein referredto as -Chol-3′), an appropriately modified solid support is used for RNAsynthesis. The modified solid support is prepared as follows:

Diethyl-2-azabutane-1,4-dicarboxylate AA

A 4.7 M aqueous solution of sodium hydroxide (50 mL) is added into astirred, ice-cooled solution of ethyl glycinate hydrochloride (32.19 g,0.23 mole) in water (50 mL). Then, ethyl acrylate (23.1 g, 0.23 mole)isis added and the mixture is stirred at room temperature untilcompletion of the reaction is ascertained by TLC. After 19 h thesolution is partitioned with dichloromethane (3×100 mL). The organiclayer is dried with anhydrous sodium sulfate, filtered and evaporated.The residue is distilled to afford AA (28.8 g, 61%).

3-{Ethoxycarbonylmethyl-[6-(9H-fluoren-9-ylmethoxycarbonyl-amino)-hexanoyl]-amino}-propionicacid ethyl ester AB

Fmoc-6-amino-hexanoic acid (9.12 g, 25.83 mmol) is dissolved indichloromethane (50 mL) and cooled with ice. Diisopropylcarbodiimde(3.25 g, 3.99 mL, 25.83 mmol) is added to the solution at 0° C. It isthen followed by the addition of Diethyl-azabutane-1,4-dicarboxylate (5g, 24.6 mmol) and dimethylamino pyridine (0.305 g, 2.5 mmol). Thesolution is brought to room temperature and stirred further for 6 h.Completion of the reaction is ascertained by TLC. The reaction mixtureis concentrated under vacuum and ethyl acetate is added to precipitatediisopropyl urea. The suspension is filtered. The filtrate is washedwith 5% aqueous hydrochloric acid, 5% sodium bicarbonate and water. Thecombined organic layer is dried over sodium sulfate and concentrated togive the crude product which is purified by column chromatography (50%EtOAC/Hexanes) to yield 11.87 g (88%) of AB.

3-[(6-Amino-hexanoyl)-ethoxycarbonylmethyl-amino]-propionic acid ethylester AC

3-{Ethoxycarbonylmethyl-[6-(9H-fluoren-9-ylmethoxycarbonylamino)-hexanoyl]-amino}-propionicacid ethyl ester AB (11.5 g, 21.3 mmol) is dissolved in 20% piperidinein dimethylformamide at 0° C. The solution is continued stirring for 1h. The reaction mixture is concentrated under vacuum, water is added tothe residue, and the product is extracted with ethyl acetate. The crudeproduct is purified by conversion into its hydrochloride salt.

3-({6-[17-(1,5-Dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yloxycarbonylamino]-hexanoyl}ethoxycarbonylmethyl-amino)-propionicacid ethyl ester AD

The hydrochloride salt of3-[(6-Amino-hexanoyl)-ethoxycarbonylmethyl-amino]-propionic acid ethylester AC (4.7 g, 14.8 mmol) is taken up in dichloromethane. Thesuspension is cooled to 0° C. on ice. To the suspensiondiisopropylethylamine (3.87 g, 5.2 mL, 30 mmol) is added. To theresulting solution cholesteryl chloroformate (6.675 g, 14.8 mmol) isadded. The reaction mixture is stirred overnight. The reaction mixtureis diluted with dichloromethane and washed with 10% hydrochloric acid.The product is purified by flash chromatography (10.3 g, 92%).

1-{6-[17-(1,5-Dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yloxycarbonylamino]-hexanoyl}-4-oxo-pyrrolidine-3-carboxylicacid ethyl ester AE

Potassium t-butoxide (1.1 g, 9.8 mmol) is slurried in 30 mL of drytoluene. The mixture is cooled to 0° C. on ice and 5 g (6.6 mmol) ofdiester AD is added slowly with stirring within 20 mins. The temperatureis kept below 5° C. during the addition. The stirring is continued for30 mins at 0° C. and 1 mL of glacial acetic acid is added, immediatelyfollowed by 4 g of NaH₂PO₄.H₂O in 40 mL of water The resultant mixtureis extracted twice with 100 mL of dichloromethane each and the combinedorganic extracts are washed twice with 10 mL of phosphate buffer each,dried, and evaporated to dryness. The residue is dissolved in 60 mL oftoluene, cooled to 0° C. and extracted with three 50 mL portions of coldpH 9.5 carbonate buffer. The aqueous extracts are adjusted to pH 3 withphosphoric acid, and extracted with five 40 mL portions of chloroformwhich are combined, dried and evaporated to dryness. The residue ispurified by column chromatography using 25% ethylacetate/hexane toafford 1.9 g of b-ketoester (39%).

[6-(3-Hydroxy-4-hydroxymethyl-pyrrolidin-1-yl)-6-oxo-hexyl]-carbamicacid17-(1,5-dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-ylester AF

Methanol (2 mL) is added dropwise over a period of 1 h to a refluxingmixture of b-ketoester AE (1.5 g, 2.2 mmol) and sodium borohydride(0.226 g, 6 mmol) in tetrahydrofuran (10 mL). Stirring is continued atreflux temperature for 1 h. After cooling to room temperature, 1 N HCl(12.5 mL) is added, the mixture is extracted with ethylacetate (3×40mL). The combined ethylacetate layer is dried over anhydrous sodiumsulfate and concentrated under vacuum to yield the product which ispurified by column chromatography (10% MeOH/CHCl₃) (89%).

(6-{3-[Bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-4-hydroxy-pyrrolidin-1-yl}-6-oxo-hexyl)-carbamicacid17-(1,5-dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-ylester AG

Diol AF (1.25 gm 1.994 mmol) is dried by evaporating with pyridine (2×5mL) in vacuo. Anhydrous pyridine (10 mL) and4,4′-dimethoxytritylchloride (0.724 g, 2.13 mmol) are added withstirring. The reaction is carried out at room temperature overnight. Thereaction is quenched by the addition of methanol. The reaction mixtureis concentrated under vacuum and to the residue dichloromethane (50 mL)is added. The organic layer is washed with 1M aqueous sodiumbicarbonate. The organic layer is dried over anhydrous sodium sulfate,filtered and concentrated. The residual pyridine is removed byevaporating with toluene. The crude product is purified by columnchromatography (2% MeOH/Chloroform, Rf=0.5 in 5% MeOH/CHCl₃) (1.75 g,95%).

Succinic acidmono-(4-[bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-1-{6-[17-(1,5-dimethyl-hexyl)-10,13-dimethyl2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1Hcyclopenta[a]phenanthren-3-yloxycarbonylamino]-hexanoyl}-pyrrolidin-3-yl)ester AH

Compound AG (1.0 g, 1.05 mmol) is mixed with succinic anhydride (0.150g, 1.5 mmol) and DMAP (0.073 g, 0.6 mmol) and dried in a vacuum at 40°C. overnight. The mixture is dissolved in anhydrous dichloroethane (3mL), triethylamine (0.318 g, 0.440 mL, 3.15 mmol) is added and thesolution is stirred at room temperature under argon atmosphere for 16 h.It is then diluted with dichloromethane (40 mL) and washed with ice coldaqueous citric acid (5 wt %, 30 mL) and water (2×20 mL). The organicphase is dried over anhydrous sodium sulfate and concentrated todryness. The residue is used as such for the next step.

Cholesterol Derivatised CPG AI

Succinate AH (0.254 g, 0.242 mmol) is dissolved in a mixture ofdichloromethane/acetonitrile (3:2, 3 mL). To that solution DMAP (0.0296g, 0.242 mmol) in acetonitrile (1.25 mL),2,2′-Dithio-bis(5-nitropyridine) (0.075 g, 0.242 mmol) inacetonitrile/dichloroethane (3:1, 1.25 mL) are added successively. Tothe resulting solution triphenylphosphine (0.064 g, 0.242 mmol) inacetonitrile (0.6 ml) is added. The reaction mixture turned brightorange in color. The solution is agitated briefly using a wrist-actionshaker (5 mins). Long chain alkyl amine-CPG (LCAA-CPG) (1.5 g, 61 mM) isadded. The suspension is agitated for 2 h. The CPG is filtered through asintered funnel and washed with acetonitrile, dichloromethane and ethersuccessively. Unreacted amino groups are masked using aceticanhydride/pyridine. The achieved loading of the CPG is measured bytaking UV measurement (37 mM/g).

The synthesis of siRNAs bearing a 5′-12-dodecanoic acid bisdecylamidegroup (herein referred to as “5′-C32-”) or a 5′-cholesteryl derivativegroup (herein referred to as “5′-Chol-”) is performed as described in WO2004/065601, except that, for the cholesteryl derivative, the oxidationstep is performed using the Beaucage reagent in order to introduce aphosphorothioate linkage at the 5′-end of the nucleic acid oligomer.

Nucleic acid sequences are represented herein using standardnomenclature, and specifically the abbreviations of Table 1.

TABLE 1 Abbreviations of nucleoside monomers used in nucleic acidsequence representation. It will be understood that these monomers, whenpresent in an oligonucleotide, are mutually linked by5′-3′-phosphodiester bonds. Abbreviation Nucleoside(s) A adenosine Ccytidine G guanosine U uridine N any nucleotide (G, A, C, U, or dT) a2′-O-methyladenosine c 2′-O-methylcytidine g 2′-O-methylguanosine u2′-O-methyluridine dT 2′-deoxythymidine s a phosphorothioate linkage

Example 2 siRNA Design and Synthesis

Transcripts

siRNA design was carried out to identify siRNAs targeting the G-alpha qsubunit (GNAQ) of a heterotrimeric G gene. Three sets were designed,each specific for a different set of cross species: 1: human and monkey;2) human, monkey and mouse; and 3) mouse and rat. GNAQ sequences wereobtained from the NCBI Refseq collection on Nov. 24, 2008 as follows:

Species GNAQ sequence ref human NM_002072.2 rat NM_031036.1 monkeyAB170509.1 mouse NM_008139.5

siRNA Design and Specificity Prediction

The predicted specificity of all possible 19mers was determined for eachsequence. The GNAQ siRNAs were used in a comprehensive search againstthe human, cynomolgous monkey, mouse and rat transcriptomes (defined asthe set of NM_(—) and XM_records within the NCBI Refseq set for human,mouse and rat, and the ‘core’ sequences from the Unigene clusters forMacaca fascicularis) using the FASTA algorithm. The Python script‘offtargetFasta.py’ was then used to parse the alignments and generate ascore based on the position and number of mismatches between the siRNAand any potential ‘off-target’ transcript. The off-target score isweighted to emphasize differences in the ‘seed’ region of siRNAs, inpositions 2-9 from the 5′ end of the molecule. The off-target score iscalculated as follows: mismatches between the oligo and the transcriptare given penalties. A mismatch in the seed region in positions 2-9 ofthe oligo is given a penalty of 2.8; mismatches in the putative cleavagesites 10 and 11 are given a penalty of 1.2, and all other mismatches apenalty of 1. The off-target score for each oligo-transcript pair isthen calculated by summing the mismatch penalties. The lowest off-targetscore from all the oligo-transcript pairs is then determined and used insubsequent sorting of oligos. Both siRNA strands were assigned to acategory of specificity according to the calculated scores: a scoreabove 3 qualifies as highly specific, equal to 3 as specific, andbetween 2.2 and 2.8 as moderately specific. In picking which oligos tosynthesize, off-target score of the antisense strand was sorted fromhigh to low.

Synthesis of dsRNA

The sense and antisense strands of the dsRNA duplexes were synthesizedon a MerMade 192 synthesizer at 1 μmol scale. For each sense andantisense sequence listed in Tables 2a, 3a, and 4a, sequence weremodified as follows and as listed in Tables 2d, 3d, and 4d:

-   -   1. In the sense strand, all pyrimidines (U, C) were replaced        with corresponding 2′-O-Methyl bases (2′ O-Methyl C and        2′-O-Methyl U); in the antisense strand, all pyrimidines (U, C)        adjacent to A (UA, CA) were replaced with corresponding        2′-O-Methyl bases (2′ O-Methyl C and 2′-O-Methyl U); a 2 base        dTdT extension at the 3′ end of both strands was introduced.    -   2. In the sense strand, all pyrimidines (U, C) are replaced with        corresponding 2′-O-Methyl bases (2′ O-Methyl C and 2′-O-Methyl        U); in the antisense strand, all pyrimidines (U, C) adjacent to        A (UA, CA) are replaced with corresponding 2′-O-Methyl bases (2′        O-Methyl C and 2′-O-Methyl U); a 2 base dTsdT (including a        phosphorothioate) extension at the 3′ end of both strands was        introduced.    -   3. In the sense strand, all pyrimidines (U, C) are replaced with        corresponding 2′-O-Methyl bases (2′ O-Methyl C and 2′-O-Methyl        U); in the antisense strand, all pyrimidines (U, C) adjacent to        A (UA, CA) and all U adjacent to another U (UU) or G (UG) were        replaced with corresponding 2′-O-Methyl bases (2′ O-Methyl C and        2′-O-Methyl U); a 2 base dTsdT (including a phosphorothioate)        extension at the 3′ end of both strands was introduced.

The synthesis of each strand of the dsRNA used solid supportedoligonucleotide synthesis using phosphoramidite chemistry.

Synthesis was performed at lumole scale in 96 well plates. The amiditesolutions were prepared at 0.1M concentration and ethyl thio tetrazole(0.6M in Acetonitrile) was used as an activator. The synthesizedsequences were cleaved and deprotected in 96 well plates, usingmethylamine in the first step and triethylamine.3HF in the second step.The crude sequences thus obtained were precipitated using acetone:ethanol mix and the pellet were re-suspended in 0.5M sodium acetatebuffer. Samples from each sequence were analyzed by LC-MS and theresulting mass data confirmed the identity of the sequences. A selectedset of samples were also analyzed by IEX chromatography.

All sequences were purified on AKTA explorer purification system usingSource 15Q column. A single peak corresponding to the full lengthsequence was collected in the eluent and was subsequently analyzed forpurity by ion exchange chromatography.

The purified sequences were desalted on a Sephadex G25 column using AKTApurifier. The desalted sequences were analyzed for concentration andpurity. For the preparation of duplexes, equimolar amounts of sense andantisense strand were heated in the required buffer (e.g. 1×PBS) at 95°C. for 2-5 minutes and slowly cooled to room temperature. Integrity ofthe duplex was confirmed by HPLC analysis.

Synthesis and Duplex Annealing for In Vivo Studies

Step 1. Oligonucleotide Synthesis

Oligonucleotides for in vivo studies were synthesized on anAKTAoligopilot synthesizer or on an ABI 394 DNA/RNA synthesizer.Commercially available controlled pore glass solid support (dT-CPG,500{acute over (Å)}, Prime Synthesis) or the in-house synthesized solidsupport cholesterol-CPG, AI were used for the synthesis. Other ligandconjugated solid supports amenable to the invention are described inU.S. patent application Ser. No. 10/946,873 filed Sep. 21, 2004, whichis hereby incorporated by reference for all purposes. RNAphosphoramidites and 2′-O-methyl modified RNA phosphoramidites withstandard protecting groups(5′-O-dimethoxytrityl-N6-benzoyl-2′-t-butyldimethylsilyl-adenosine-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramidite,5′-O-dimethoxytrityl-N4-acetyl-2′-t-butyldimethylsilyl-cytidine-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramidite,5′-O-dimethoxytrityl-N2-isobutryl-2′-t-butyldimethylsilyl-guanosine-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramidite,5′-O-dimethoxytrityl-2′-t-butyldimethylsilyl-uridine-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramidite,5′-O-dimethoxytrityl-N6-benzoyl-2′-O-methyl-adenosine-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramidite,5′-O-dimethoxytrityl-N4-acetyl-2′-O-methyl-cytidine-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramidite,5′-O-dimethoxytrityl-N2-isobutryl-2′-O-methyl-guanosine-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramidite,5′-O-dimethoxytrityl-2′-O-methyl-uridine-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramiditeand5′-O-dimethoxytrityl-2′-deoxy-thymidine-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramidite)were obtained commercially (e.g. from Pierce Nucleic Acids Technologiesand ChemGenes Research).

For the syntheses on AKTAoligopilot synthesizer, all phosphoramiditeswere used at a concentration of 0.2 M in CH₃CN except for guanosine and2′-O-methyl-uridine, which were used at 0.2 M concentration in 10%THF/CH₃CN (v/v). Coupling/recycling time of 16 minutes was used for allphosphoramidite couplings. The activator was 5-ethyl-thio-tetrazole(0.75 M, American International Chemicals). For the PO-oxidation, 50 mMiodine in water/pyridine (10:90 v/v) was used and for the PS-oxidation2% PADS (GL Synthesis) in 2,6-lutidine/CH₃CN (1:1 v/v) was used. For thesyntheses on ABI 394 DNA/RNA synthesizer, all phosphoramidites were usedat a concentration of 0.15 M in CH₃CN except for 2′-O-methyl-uridine,which was used at 0.15 M concentration in 10% THF/CH₃CN (v/v). Couplingtime of 10 minutes was used for all phosphoramidite couplings. Theactivator was 5-ethyl-thio-tetrazole (0.25 M, Glen Research). For thePO-oxidation, 20 mM iodine in water/pyridine (Glen Research) was usedand for the PS-oxidation 0.1M DDTT (AM Chemicals) in pyridine was used.

Step 2. Deprotection of Oligonucleotides

After completion of synthesis, the support was transferred to a 100 mLglass bottle (VWR). The oligonucleotide was cleaved from the supportwith simultaneous deprotection of base and phosphate groups with 40 mLof a 40% aq. methyl amine (Aldrich) 90 mins at 45° C. The bottle wascooled briefly on ice and then the methylamine was filtered into a new500 mL bottle. The CPG was washed three times with 40 mL portions ofDMSO. The mixture was then cooled on dry ice.

In order to remove the tert-butyldimethylsilyl (TBDMS) groups at the 2′position, 60 mL triethylamine trihydrofluoride (Et3N—HF) was added tothe above mixture. The mixture was heated at 40° C. for 60 minutes. Thereaction was then quenched with 220 mL of 50 mM sodium acetate (pH 5.5)and stored in the freezer until purification.

Sequences Synthesized on the ABI DNA/RNA Synthesizer

After completion of synthesis, the support was transferred to a 15 mLtube (VWR). The oligonucleotide was cleaved from the support withsimultaneous deprotection of base and phosphate groups with 7 mL of a40% aq. methyl amine (Aldrich) 15 mins at 65° C. The bottle was cooledbriefly on ice and then the methylamine solution was filtered into a 100mL bottle (VWR). The CPG was washed three times with 7 mL portions ofDMSO. The mixture was then cooled on dry ice.

In order to remove the tert-butyldimethylsilyl (TBDMS) groups at the 2′position, 10.5 mL triethylamine trihydrofluoride (Et3N—HF) was added tothe above mixture. The mixture was heated at 60° C. for 15 minutes. Thereaction was then quenched with 38.5 mL of 50 mM sodium acetate (pH 5.5)and stored in the freezer until purification.

Step 3. Quantitation of Crude Oligonucleotides For all samples, a 10 μLaliquot was diluted with 990 μL of deionised nuclease free water (1.0mL) and the absorbance reading at 260 nm obtained.

Step 4. Purification of Oligonucleotides

Unconjugated Oligonucleotides

The unconjugated samples were purified by HPLC on a TSK-Gel SuperQ-5PW(20) column packed in house (17.3×5 cm) or on a commercially availableTSK-Gel SuperQ-5PW column (15×0.215 cm) available from TOSOH Bioscience.The buffers were 20 mM phosphate in 10% CH₃CN, pH 8.5 (buffer A) and 20mM phosphate, 1.0 M NaBr in 10% CH₃CN, pH 8.5 (buffer B). The flow ratewas 50.0 mL/min for the in house packed column and 10.0 ml/min for thecommercially obtained column. Wavelengths of 260 and 294 nm weremonitored. The fractions containing the full-length oligonucleotideswere pooled together, evaporated, and reconstituted to ˜100 mL withdeionised water.

Cholesterol-conjugated Oligonucleotides

The cholesterol conjugated sequences were HPLC purified on RPC-Source 15reverse-phase columns packed in house (17.3×5 cm or 15×2 cm). Thebuffers were 20 mM NaOAc in 10% CH₃CN (buffer A) and 20 mM NaOAc in 70%CH₃CN (buffer B). The flow rate was 50.0 mL/min for the 17.3×5 cm columnand 12.0 ml/min for the 15×2 cm column. Wavelengths of 260 and 284 nmwere monitored. The fractions containing the full-lengtholigonucleotides were pooled, evaporated, and reconstituted to 100 mLwith deionised water.

Step 5. Desalting of Purified Oligonucleotides

The purified oligonucleotides were desalted on either an AKTA Exploreror an AKTA Prime system (Amersham Biosciences) using a Sephadex G-25column packed in house. First, the column was washed with water at aflow rate of 40 mL/min for 20-30 min. The sample was then applied in40-60 mL fractions. The eluted salt-free fractions were combined, dried,and reconstituted in ˜50 mL of RNase free water.

Step 6. Purity Analysis

Approximately 0.3 OD of each of the desalted oligonucleotides wasdiluted in water to 3004 and were analyzed by CGE, ion exchange HPLC,and LC/MS.

Step 7. Duplex Formation

For the preparation of duplexes, equimolar amounts of sense andantisense strand were heated in the required buffer (e.g. 1×PBS) at 95°C. for 5 min and slowly cooled to room temperature. Integrity of theduplex was confirmed by HPLC analysis.

Tables of dsRNA Sequences

Table 2 provides sequences used for design of dsRNA targeting human GNAQthat will cross react with monkey GNAQ. Table 3 provides sequences usedfor design of dsRNA targeting human GNAQ that will cross react with bothmonkey and rat GNAQ. Table 4 provides sequences used for design of dsRNAtargeting rat GNAQ that will cross react with mouse GNAQ.

Tables 2a, 3a, and 4a following tables provide the sense and antisensestrand of GNAQ target sequences. Tables 2b, 3b, and 4b provide exemplarysense and antisense dsRNA strands with a NN 2 base overhang. Tables 2c,3c, and 4c provide exemplary sense and antisense dsRNA strands with dTdT2 base overhang. Tables 2d, 3d, and 4d provide sequences of dsRNA thatwere synthesized, including the dTdT 2 base overhang and modifiednucleotides.

TABLE 2a GNAQ (human X monkey): target sequences Numbering for targetsequences is based on Human GNAQ NM_002072. Start of SEQ SEQ target IDTarget sequence, sense ID Target sequence, antisense sequence NO. strand(5′-3′) NO. strand (5′-3′) 1217 1 CUAAUUUAUUGCCGUCCUG 74CAGGACGGCAAUAAAUUAG 1213 2 AAUACUAAUUUAUUGCCGU 75 ACGGCAAUAAAUUAGUAUU1810 3 CAGCCAUAGCUUGAUUGCU 76 AGCAAUCAAGCUAUGGCUG 1590 4GUCAGGACACAUCGUUCGA 77 UCGAACGAUGUGUCCUGAC 1149 5 CUUCCCUGGUGGGCUAUUG 78CAAUAGCCCACCAGGGAAG 1971 6 GACACUACAUUACCCUAAU 79 AUUAGGGUAAUGUAGUGUC1237 7 ACUCUGUGUGAGCGUGUCC 80 GGACACGCUCACACAGAGU 1152 8CCCUGGUGGGCUAUUGAAG 81 CUUCAAUAGCCCACCAGGG 1216 9 ACUAAUUUAUUGCCGUCCU 82AGGACGGCAAUAAAUUAGU 1575 10 CUCUCAAAUGAUACAGUCA 83 UGACUGUAUCAUUUGAGAG1105 11 AGUACAAUCUGGUCUAAUU 84 AAUUAGACCAGAUUGUACU 1407 12CACAAAGAUAAGACUUGUU 85 AACAAGUCUUAUCUUUGUG 1108 13 ACAAUCUGGUCUAAUUGUG86 CACAAUUAGACCAGAUUGU 1395 14 CAGUCAUGCACUCACAAAG 87CUUUGUGAGUGCAUGACUG 1595 15 GACACAUCGUUCGAUUUAA 88 UUAAAUCGAACGAUGUGUC1992 16 CUGCUACCCAGAACCUUUU 89 AAAAGGUUCUGGGUAGCAG 1809 17UCAGCCAUAGCUUGAUUGC 90 GCAAUCAAGCUAUGGCUGA 1220 18 AUUUAUUGCCGUCCUGGAC91 GUCCAGGACGGCAAUAAAU 1203 19 CAAUUUGCAUAAUACUAAU 92AUUAGUAUUAUGCAAAUUG 1322 20 GUACAGUCCCAGCACAUUU 93 AAAUGUGCUGGGACUGUAC1804 21 UACCUUCAGCCAUAGCUUG 94 CAAGCUAUGGCUGAAGGUA 1968 22ACAGACACUACAUUACCCU 95 AGGGUAAUGUAGUGUCUGU 1214 23 AUACUAAUUUAUUGCCGUC96 GACGGCAAUAAAUUAGUAU 1159 24 GGGCUAUUGAAGAUACACA 97UGUGUAUCUUCAAUAGCCC 1603 25 GUUCGAUUUAAGCCAUCAU 98 AUGAUGGCUUAAAUCGAAC1123 26 UGUGCCUCCUAGACACCCG 99 CGGGUGUCUAGGAGGCACA 1233 27CUGGACUCUGUGUGAGCGU 100 ACGCUCACACAGAGUCCAG 1930 28 ACCCUCUCUUUCAAUUGCA101 UGCAAUUGAAAGAGAGGGU 1969 29 CAGACACUACAUUACCCUA 102UAGGGUAAUGUAGUGUCUG 1219 30 AAUUUAUUGCCGUCCUGGA 103 UCCAGGACGGCAAUAAAUU1241 31 UGUGUGAGCGUGUCCACAG 104 CUGUGGACACGCUCACACA 1153 32CCUGGUGGGCUAUUGAAGA 105 UCUUCAAUAGCCCACCAGG 1805 33 ACCUUCAGCCAUAGCUUGA106 UCAAGCUAUGGCUGAAGGU 1312 34 GGAUGCUGAAGUACAGUCC 107GGACUGUACUUCAGCAUCC 1546 35 AUCCUAGUUCCAUUCUUGG 108 CCAAGAAUGGAACUAGGAU1547 36 UCCUAGUUCCAUUCUUGGU 109 ACCAAGAAUGGAACUAGGA 1103 37GGAGUACAAUCUGGUCUAA 110 UUAGACCAGAUUGUACUCC 1334 38 CACAUUUCCUCUCUAUCUU111 AAGAUAGAGAGGAAAUGUG 1255 39 CACAGAGUUUGUAGUAAAU 112AUUUACUACAAACUCUGUG 1967 40 AACAGACACUACAUUACCC 113 GGGUAAUGUAGUGUCUGUU1391 41 UUCUCAGUCAUGCACUCAC 114 GUGAGUGCAUGACUGAGAA 1124 42GUGCCUCCUAGACACCCGC 115 GCGGGUGUCUAGGAGGCAC 1612 43 AAGCCAUCAUCAGCUUAAU116 AUUAAGCUGAUGAUGGCUU 1933 44 CUCUCUUUCAAUUGCAGAU 117AUCUGCAAUUGAAAGAGAG 1078 45 ACACCAUCCUCCAGUUGAA 118 UUCAACUGGAGGAUGGUGU1545 46 UAUCCUAGUUCCAUUCUUG 119 CAAGAAUGGAACUAGGAUA 1109 47CAAUCUGGUCUAAUUGUGC 120 GCACAAUUAGACCAGAUUG 1398 48 UCAUGCACUCACAAAGAUA121 UAUCUUUGUGAGUGCAUGA 1970 49 AGACACUACAUUACCCUAA 122UUAGGGUAAUGUAGUGUCU 1173 50 ACACAAGAGGGACUGUAUU 123 AAUACAGUCCCUCUUGUGU1313 51 GAUGCUGAAGUACAGUCCC 124 GGGACUGUACUUCAGCAUC 1811 52AGCCAUAGCUUGAUUGCUC 125 GAGCAAUCAAGCUAUGGCU 1862 53 CACAGGAGUCCUUUCUUUU126 AAAAGAAAGGACUCCUGUG 1600 54 AUCGUUCGAUUUAAGCCAU 127AUGGCUUAAAUCGAACGAU 1618 55 UCAUCAGCUUAAUUUAAGU 128 ACUUAAAUUAAGCUGAUGA1332 56 AGCACAUUUCCUCUCUAUC 129 GAUAGAGAGGAAAUGUGCU 1157 57GUGGGCUAUUGAAGAUACA 130 UGUAUCUUCAAUAGCCCAC 888 58 AUCAUGUAUUCCCAUCUAG131 CUAGAUGGGAAUACAUGAU 1855 59 AAAGACACACAGGAGUCCU 132AGGACUCCUGUGUGUCUUU 1579 60 CAAAUGAUACAGUCAGGAC 133 GUCCUGACUGUAUCAUUUG805 61 UUAGAACAAUUAUCACAUA 134 UAUGUGAUAAUUGUUCUAA 1554 62UCCAUUCUUGGUCAAGUUU 135 AAACUUGACCAAGAAUGGA 1113 63 CUGGUCUAAUUGUGCCUCC136 GGAGGCACAAUUAGACCAG 1174 64 CACAAGAGGGACUGUAUUU 137AAAUACAGUCCCUCUUGUG 1735 65 UCUUGUCUCACUUUGGACU 138 AGUCCAAAGUGAGACAAGA1450 66 UUUUCUAUGGAGCAAAACA 139 UGUUUUGCUCCAUAGAAAA 1285 67AUUUAAACUAUUCAGAGGA 140 UCCUCUGAAUAGUUUAAAU 804 68 UUUAGAACAAUUAUCACAU141 AUGUGAUAAUUGUUCUAAA 1866 69 GGAGUCCUUUCUUUUGAAA 142UUUCAAAAGAAAGGACUCC 1610 70 UUAAGCCAUCAUCAGCUUA 143 UAAGCUGAUGAUGGCUUAA1117 71 UCUAAUUGUGCCUCCUAGA 144 UCUAGGAGGCACAAUUAGA 1320 72AAGUACAGUCCCAGCACAU 145 AUGUGCUGGGACUGUACUU 1317 73 CUGAAGUACAGUCCCAGCA146 UGCUGGGACUGUACUUCAG

TABLE 2b GNAQ (human and monkey): sense and antisense sequences with 2base overhangs; Numbering for target sequences is based on Human GNAQNM_002072. Start of target SEQ ID NO SEQUENCE (5′-3′) Strand sequence147 CUAAUUUAUUGCCGUCCUGNN sense 1217 148 CAGGACGGCAAUAAAUUAGNN antis1217 149 AAUACUAAUUUAUUGCCGUNN sense 1213 150 ACGGCAAUAAAUUAGUAUUNNantis 1213 151 CAGCCAUAGCUUGAUUGCUNN sense 1810 152AGCAAUCAAGCUAUGGCUGNN antis 1810 153 GUCAGGACACAUCGUUCGANN sense 1590154 UCGAACGAUGUGUCCUGACNN antis 1590 155 CUUCCCUGGUGGGCUAUUGNN sense1149 156 CAAUAGCCCACCAGGGAAGNN antis 1149 157 GACACUACAUUACCCUAAUNNsense 1971 158 AUUAGGGUAAUGUAGUGUCNN antis 1971 159ACUCUGUGUGAGCGUGUCCNN sense 1237 160 GGACACGCUCACACAGAGUNN antis 1237161 CCCUGGUGGGCUAUUGAAGNN sense 1152 162 CUUCAAUAGCCCACCAGGGNN antis1152 163 ACUAAUUUAUUGCCGUCCUNN sense 1216 164 AGGACGGCAAUAAAUUAGUNNantis 1216 165 CUCUCAAAUGAUACAGUCANN sense 1575 166UGACUGUAUCAUUUGAGAGNN antis 1575 167 AGUACAAUCUGGUCUAAUUNN sense 1105168 AAUUAGACCAGAUUGUACUNN antis 1105 169 CACAAAGAUAAGACUUGUUNN sense1407 170 AACAAGUCUUAUCUUUGUGNN antis 1407 171 ACAAUCUGGUCUAAUUGUGNNsense 1108 172 CACAAUUAGACCAGAUUGUNN antis 1108 173CAGUCAUGCACUCACAAAGNN sense 1395 174 CUUUGUGAGUGCAUGACUGNN antis 1395175 GACACAUCGUUCGAUUUAANN sense 1595 176 UUAAAUCGAACGAUGUGUCNN antis1595 177 CUGCUACCCAGAACCUUUUNN sense 1992 178 AAAAGGUUCUGGGUAGCAGNNantis 1992 179 UCAGCCAUAGCUUGAUUGCNN sense 1809 180GCAAUCAAGCUAUGGCUGANN antis 1809 181 AUUUAUUGCCGUCCUGGACNN sense 1220182 GUCCAGGACGGCAAUAAAUNN antis 1220 183 CAAUUUGCAUAAUACUAAUNN sense1203 184 AUUAGUAUUAUGCAAAUUGNN antis 1203 185 GUACAGUCCCAGCACAUUUNNsense 1322 186 AAAUGUGCUGGGACUGUACNN antis 1322 187UACCUUCAGCCAUAGCUUGNN sense 1804 188 CAAGCUAUGGCUGAAGGUANN antis 1804189 ACAGACACUACAUUACCCUNN sense 1968 190 AGGGUAAUGUAGUGUCUGUNN antis1968 191 AUACUAAUUUAUUGCCGUCNN sense 1214 192 GACGGCAAUAAAUUAGUAUNNantis 1214 193 GGGCUAUUGAAGAUACACANN sense 1159 194UGUGUAUCUUCAAUAGCCCNN antis 1159 195 GUUCGAUUUAAGCCAUCAUNN sense 1603196 AUGAUGGCUUAAAUCGAACNN antis 1603 197 UGUGCCUCCUAGACACCCGNN sense1123 198 CGGGUGUCUAGGAGGCACANN antis 1123 199 CUGGACUCUGUGUGAGCGUNNsense 1233 200 ACGCUCACACAGAGUCCAGNN antis 1233 201ACCCUCUCUUUCAAUUGCANN sense 1930 202 UGCAAUUGAAAGAGAGGGUNN antis 1930203 CAGACACUACAUUACCCUANN sense 1969 204 UAGGGUAAUGUAGUGUCUGNN antis1969 205 AAUUUAUUGCCGUCCUGGANN sense 1219 206 UCCAGGACGGCAAUAAAUUNNantis 1219 207 UGUGUGAGCGUGUCCACAGNN sense 1241 208CUGUGGACACGCUCACACANN antis 1241 209 CCUGGUGGGCUAUUGAAGANN sense 1153210 UCUUCAAUAGCCCACCAGGNN antis 1153 211 ACCUUCAGCCAUAGCUUGANN sense1805 212 UCAAGCUAUGGCUGAAGGUNN antis 1805 213 GGAUGCUGAAGUACAGUCCNNsense 1312 214 GGACUGUACUUCAGCAUCCNN antis 1312 215AUCCUAGUUCCAUUCUUGGNN sense 1546 216 CCAAGAAUGGAACUAGGAUNN antis 1546217 UCCUAGUUCCAUUCUUGGUNN sense 1547 218 ACCAAGAAUGGAACUAGGANN antis1547 219 GGAGUACAAUCUGGUCUAANN sense 1103 220 UUAGACCAGAUUGUACUCCNNantis 1103 221 CACAUUUCCUCUCUAUCUUNN sense 1334 222AAGAUAGAGAGGAAAUGUGNN antis 1334 223 CACAGAGUUUGUAGUAAAUNN sense 1255224 AUUUACUACAAACUCUGUGNN antis 1255 225 AACAGACACUACAUUACCCNN sense1967 226 GGGUAAUGUAGUGUCUGUUNN antis 1967 227 UUCUCAGUCAUGCACUCACNNsense 1391 228 GUGAGUGCAUGACUGAGAANN antis 1391 229GUGCCUCCUAGACACCCGCNN sense 1124 230 GCGGGUGUCUAGGAGGCACNN antis 1124231 AAGCCAUCAUCAGCUUAAUNN sense 1612 232 AUUAAGCUGAUGAUGGCUUNN antis1612 233 CUCUCUUUCAAUUGCAGAUNN sense 1933 234 AUCUGCAAUUGAAAGAGAGNNantis 1933 235 ACACCAUCCUCCAGUUGAANN sense 1078 236UUCAACUGGAGGAUGGUGUNN antis 1078 237 UAUCCUAGUUCCAUUCUUGNN sense 1545238 CAAGAAUGGAACUAGGAUANN antis 1545 239 CAAUCUGGUCUAAUUGUGCNN sense1109 240 GCACAAUUAGACCAGAUUGNN antis 1109 241 UCAUGCACUCACAAAGAUANNsense 1398 242 UAUCUUUGUGAGUGCAUGANN antis 1398 243AGACACUACAUUACCCUAANN sense 1970 244 UUAGGGUAAUGUAGUGUCUNN antis 1970245 ACACAAGAGGGACUGUAUUNN sense 1173 246 AAUACAGUCCCUCUUGUGUNN antis1173 247 GAUGCUGAAGUACAGUCCCNN sense 1313 248 GGGACUGUACUUCAGCAUCNNantis 1313 249 AGCCAUAGCUUGAUUGCUCNN sense 1811 250GAGCAAUCAAGCUAUGGCUNN antis 1811 251 CACAGGAGUCCUUUCUUUUNN sense 1862252 AAAAGAAAGGACUCCUGUGNN antis 1862 253 AUCGUUCGAUUUAAGCCAUNN sense1600 254 AUGGCUUAAAUCGAACGAUNN antis 1600 255 UCAUCAGCUUAAUUUAAGUNNsense 1618 256 ACUUAAAUUAAGCUGAUGANN antis 1618 257AGCACAUUUCCUCUCUAUCNN sense 1332 258 GAUAGAGAGGAAAUGUGCUNN antis 1332259 GUGGGCUAUUGAAGAUACANN sense 1157 260 UGUAUCUUCAAUAGCCCACNN antis1157 261 AUCAUGUAUUCCCAUCUAGNN sense 888 262 CUAGAUGGGAAUACAUGAUNN antis888 263 AAAGACACACAGGAGUCCUNN sense 1855 264 AGGACUCCUGUGUGUCUUUNN antis1855 265 CAAAUGAUACAGUCAGGACNN sense 1579 266 GUCCUGACUGUAUCAUUUGNNantis 1579 267 UUAGAACAAUUAUCACAUANN sense 805 268 UAUGUGAUAAUUGUUCUAANNantis 805 269 UCCAUUCUUGGUCAAGUUUNN sense 1554 270 AAACUUGACCAAGAAUGGANNantis 1554 271 CUGGUCUAAUUGUGCCUCCNN sense 1113 272GGAGGCACAAUUAGACCAGNN antis 1113 273 CACAAGAGGGACUGUAUUUNN sense 1174274 AAAUACAGUCCCUCUUGUGNN antis 1174 275 UCUUGUCUCACUUUGGACUNN sense1735 276 AGUCCAAAGUGAGACAAGANN antis 1735 277 UUUUCUAUGGAGCAAAACANNsense 1450 278 UGUUUUGCUCCAUAGAAAANN antis 1450 279AUUUAAACUAUUCAGAGGANN sense 1285 280 UCCUCUGAAUAGUUUAAAUNN antis 1285281 UUUAGAACAAUUAUCACAUNN sense 804 282 AUGUGAUAAUUGUUCUAAANN antis 804283 GGAGUCCUUUCUUUUGAAANN sense 1866 284 UUUCAAAAGAAAGGACUCCNN antis1866 285 UUAAGCCAUCAUCAGCUUANN sense 1610 286 UAAGCUGAUGAUGGCUUAANNantis 1610 287 UCUAAUUGUGCCUCCUAGANN sense 1117 288UCUAGGAGGCACAAUUAGANN antis 1117 289 AAGUACAGUCCCAGCACAUNN sense 1320290 AUGUGCUGGGACUGUACUUNN antis 1320 291 CUGAAGUACAGUCCCAGCANN sense1317 292 UGCUGGGACUGUACUUCAGNN antis 1317

TABLE 2c GNAQ (human and monkey): sense and antisense sequences withdTdT overhangs Numbering for target sequences is based on Human GNAQNM_002072 Start of target SEQ ID NO SEQUENCE (5′-3′) Strand sequence 293CUAAUUUAUUGCCGUCCUGdTdT sense 1217 294 CAGGACGGCAAUAAAUUAGdTdT antis1217 295 AAUACUAAUUUAUUGCCGUdTdT sense 1213 296 ACGGCAAUAAAUUAGUAUUdTdTantis 1213 297 CAGCCAUAGCUUGAUUGCUdTdT sense 1810 298AGCAAUCAAGCUAUGGCUGdTdT antis 1810 299 GUCAGGACACAUCGUUCGAdTdT sense1590 300 UCGAACGAUGUGUCCUGACdTdT antis 1590 301 CUUCCCUGGUGGGCUAUUGdTdTsense 1149 302 CAAUAGCCCACCAGGGAAGdTdT antis 1149 303GACACUACAUUACCCUAAUdTdT sense 1971 304 AUUAGGGUAAUGUAGUGUCdTdT antis1971 305 ACUCUGUGUGAGCGUGUCCdTdT sense 1237 306 GGACACGCUCACACAGAGUdTdTantis 1237 307 CCCUGGUGGGCUAUUGAAGdTdT sense 1152 308CUUCAAUAGCCCACCAGGGdTdT antis 1152 309 ACUAAUUUAUUGCCGUCCUdTdT sense1216 310 AGGACGGCAAUAAAUUAGUdTdT antis 1216 311 CUCUCAAAUGAUACAGUCAdTdTsense 1575 312 UGACUGUAUCAUUUGAGAGdTdT antis 1575 313AGUACAAUCUGGUCUAAUUdTdT sense 1105 314 AAUUAGACCAGAUUGUACUdTdT antis1105 315 CACAAAGAUAAGACUUGUUdTdT sense 1407 316 AACAAGUCUUAUCUUUGUGdTdTantis 1407 317 ACAAUCUGGUCUAAUUGUGdTdT sense 1108 318CACAAUUAGACCAGAUUGUdTdT antis 1108 319 CAGUCAUGCACUCACAAAGdTdT sense1395 320 CUUUGUGAGUGCAUGACUGdTdT antis 1395 321 GACACAUCGUUCGAUUUAAdTdTsense 1595 322 UUAAAUCGAACGAUGUGUCdTdT antis 1595 323CUGCUACCCAGAACCUUUUdTdT sense 1992 324 AAAAGGUUCUGGGUAGCAGdTdT antis1992 325 UCAGCCAUAGCUUGAUUGCdTdT sense 1809 326 GCAAUCAAGCUAUGGCUGAdTdTantis 1809 327 AUUUAUUGCCGUCCUGGACdTdT sense 1220 328GUCCAGGACGGCAAUAAAUdTdT antis 1220 329 CAAUUUGCAUAAUACUAAUdTdT sense1203 330 AUUAGUAUUAUGCAAAUUGdTdT antis 1203 331 GUACAGUCCCAGCACAUUUdTdTsense 1322 332 AAAUGUGCUGGGACUGUACdTdT antis 1322 333UACCUUCAGCCAUAGCUUGdTdT sense 1804 334 CAAGCUAUGGCUGAAGGUAdTdT antis1804 335 ACAGACACUACAUUACCCUdTdT sense 1968 336 AGGGUAAUGUAGUGUCUGUdTdTantis 1968 337 AUACUAAUUUAUUGCCGUCdTdT sense 1214 338GACGGCAAUAAAUUAGUAUdTdT antis 1214 339 GGGCUAUUGAAGAUACACAdTdT sense1159 340 UGUGUAUCUUCAAUAGCCCdTdT antis 1159 341 GUUCGAUUUAAGCCAUCAUdTdTsense 1603 342 AUGAUGGCUUAAAUCGAACdTdT antis 1603 343UGUGCCUCCUAGACACCCGdTdT sense 1123 344 CGGGUGUCUAGGAGGCACAdTdT antis1123 345 CUGGACUCUGUGUGAGCGUdTdT sense 1233 346 ACGCUCACACAGAGUCCAGdTdTantis 1233 347 ACCCUCUCUUUCAAUUGCAdTdT sense 1930 348UGCAAUUGAAAGAGAGGGUdTdT antis 1930 349 CAGACACUACAUUACCCUAdTdT sense1969 350 UAGGGUAAUGUAGUGUCUGdTdT antis 1969 351 AAUUUAUUGCCGUCCUGGAdTdTsense 1219 352 UCCAGGACGGCAAUAAAUUdTdT antis 1219 353UGUGUGAGCGUGUCCACAGdTdT sense 1241 354 CUGUGGACACGCUCACACAdTdT antis1241 355 CCUGGUGGGCUAUUGAAGAdTdT sense 1153 356 UCUUCAAUAGCCCACCAGGdTdTantis 1153 357 ACCUUCAGCCAUAGCUUGAdTdT sense 1805 358UCAAGCUAUGGCUGAAGGUdTdT antis 1805 359 GGAUGCUGAAGUACAGUCCdTdT sense1312 360 GGACUGUACUUCAGCAUCCdTdT antis 1312 361 AUCCUAGUUCCAUUCUUGGdTdTsense 1546 362 CCAAGAAUGGAACUAGGAUdTdT antis 1546 363UCCUAGUUCCAUUCUUGGUdTdT sense 1547 364 ACCAAGAAUGGAACUAGGAdTdT antis1547 365 GGAGUACAAUCUGGUCUAAdTdT sense 1103 366 UUAGACCAGAUUGUACUCCdTdTantis 1103 367 CACAUUUCCUCUCUAUCUUdTdT sense 1334 368AAGAUAGAGAGGAAAUGUGdTdT antis 1334 369 CACAGAGUUUGUAGUAAAUdTdT sense1255 370 AUUUACUACAAACUCUGUGdTdT antis 1255 371 AACAGACACUACAUUACCCdTdTsense 1967 372 GGGUAAUGUAGUGUCUGUUdTdT antis 1967 373UUCUCAGUCAUGCACUCACdTdT sense 1391 374 GUGAGUGCAUGACUGAGAAdTdT antis1391 375 GUGCCUCCUAGACACCCGCdTdT sense 1124 376 GCGGGUGUCUAGGAGGCACdTdTantis 1124 377 AAGCCAUCAUCAGCUUAAUdTdT sense 1612 378AUUAAGCUGAUGAUGGCUUdTdT antis 1612 379 CUCUCUUUCAAUUGCAGAUdTdT sense1933 380 AUCUGCAAUUGAAAGAGAGdTdT antis 1933 381 ACACCAUCCUCCAGUUGAAdTdTsense 1078 382 UUCAACUGGAGGAUGGUGUdTdT antis 1078 383UAUCCUAGUUCCAUUCUUGdTdT sense 1545 384 CAAGAAUGGAACUAGGAUAdTdT antis1545 385 CAAUCUGGUCUAAUUGUGCdTdT sense 1109 386 GCACAAUUAGACCAGAUUGdTdTantis 1109 387 UCAUGCACUCACAAAGAUAdTdT sense 1398 388UAUCUUUGUGAGUGCAUGAdTdT antis 1398 389 AGACACUACAUUACCCUAAdTdT sense1970 390 UUAGGGUAAUGUAGUGUCUdTdT antis 1970 391 ACACAAGAGGGACUGUAUUdTdTsense 1173 392 AAUACAGUCCCUCUUGUGUdTdT antis 1173 393GAUGCUGAAGUACAGUCCCdTdT sense 1313 394 GGGACUGUACUUCAGCAUCdTdT antis1313 395 AGCCAUAGCUUGAUUGCUCdTdT sense 1811 396 GAGCAAUCAAGCUAUGGCUdTdTantis 1811 397 CACAGGAGUCCUUUCUUUUdTdT sense 1862 398AAAAGAAAGGACUCCUGUGdTdT antis 1862 399 AUCGUUCGAUUUAAGCCAUdTdT sense1600 400 AUGGCUUAAAUCGAACGAUdTdT antis 1600 401 UCAUCAGCUUAAUUUAAGUdTdTsense 1618 402 ACUUAAAUUAAGCUGAUGAdTdT antis 1618 403AGCACAUUUCCUCUCUAUCdTdT sense 1332 404 GAUAGAGAGGAAAUGUGCUdTdT antis1332 405 GUGGGCUAUUGAAGAUACAdTdT sense 1157 406 UGUAUCUUCAAUAGCCCACdTdTantis 1157 407 AUCAUGUAUUCCCAUCUAGdTdT sense 888 408CUAGAUGGGAAUACAUGAUdTdT antis 888 409 AAAGACACACAGGAGUCCUdTdT sense 1855410 AGGACUCCUGUGUGUCUUUdTdT antis 1855 411 CAAAUGAUACAGUCAGGACdTdT sense1579 412 GUCCUGACUGUAUCAUUUGdTdT antis 1579 413 UUAGAACAAUUAUCACAUAdTdTsense 805 414 UAUGUGAUAAUUGUUCUAAdTdT antis 805 415UCCAUUCUUGGUCAAGUUUdTdT sense 1554 416 AAACUUGACCAAGAAUGGAdTdT antis1554 417 CUGGUCUAAUUGUGCCUCCdTdT sense 1113 418 GGAGGCACAAUUAGACCAGdTdTantis 1113 419 CACAAGAGGGACUGUAUUUdTdT sense 1174 420AAAUACAGUCCCUCUUGUGdTdT antis 1174 421 UCUUGUCUCACUUUGGACUdTdT sense1735 422 AGUCCAAAGUGAGACAAGAdTdT antis 1735 423 UUUUCUAUGGAGCAAAACAdTdTsense 1450 424 UGUUUUGCUCCAUAGAAAAdTdT antis 1450 425AUUUAAACUAUUCAGAGGAdTdT sense 1285 426 UCCUCUGAAUAGUUUAAAUdTdT antis1285 427 UUUAGAACAAUUAUCACAUdTdT sense 804 428 AUGUGAUAAUUGUUCUAAAdTdTantis 804 429 GGAGUCCUUUCUUUUGAAAdTdT sense 1866 430UUUCAAAAGAAAGGACUCCdTdT antis 1866 431 UUAAGCCAUCAUCAGCUUAdTdT sense1610 432 UAAGCUGAUGAUGGCUUAAdTdT antis 1610 433 UCUAAUUGUGCCUCCUAGAdTdTsense 1117 434 UCUAGGAGGCACAAUUAGAdTdT antis 1117 435AAGUACAGUCCCAGCACAUdTdT sense 1320 436 AUGUGCUGGGACUGUACUUdTdT antis1320 437 CUGAAGUACAGUCCCAGCAdTdT sense 1317 438 UGCUGGGACUGUACUUCAGdTdTantis 1317

TABLE 2d GNAQ (human and monkey): modified sense and antisense strandsNumbering for target sequences is based on Human GNAQ NM_002072. Startof target SEQUENCE (5′-3′) Strand sequence SEQ ID NO: Modifications:Sense strand - all pyrimidines (U, C) are 2′OMe; antisense strand -pyrimidines adjacent to A (UA, CA) are 2′Ome; 3′end is dTdTcuAAuuuAuuGccGuccuGdTdT sense 1217 439 cAGGACGGcAAuAAAUuAGdTdT antis1217 440 AAuAcuAAuuuAuuGccGudTdT sense 1213 441 ACGGcAAuAAAUuAGuAUUdTdTantis 1213 442 cAGccAuAGcuuGAuuGcudTdT sense 1810 443AGcAAUcAAGCuAUGGCUGdTdT antis 1810 444 GucAGGAcAcAucGuucGAdTdT sense1590 445 UCGAACGAUGUGUCCUGACdTdT antis 1590 446 cuucccuGGuGGGcuAuuGdTdTsense 1149 447 cAAuAGCCcACcAGGGAAGdTdT antis 1149 448GAcAcuAcAuuAcccuAAudTdT sense 1971 449 AUuAGGGuAAUGuAGUGUCdTdT antis1971 450 AcucuGuGuGAGcGuGuccdTdT sense 1237 451 GGAcACGCUcAcAcAGAGUdTdTantis 1237 452 cccuGGuGGGcuAuuGAAGdTdT sense 1152 453CUUcAAuAGCCcACcAGGGdTdT antis 1152 454 AcuAAuuuAuuGccGuccudTdT sense1216 455 AGGACGGcAAuAAAUuAGUdTdT antis 1216 456 cucucAAAuGAuAcAGucAdTdTsense 1575 457 UGACUGuAUcAUUUGAGAGdTdT antis 1575 458AGuAcAAucuGGucuAAuudTdT sense 1105 459 AAUuAGACcAGAUUGuACUdTdT antis1105 460 cAcAAAGAuAAGAcuuGuudTdT sense 1407 461 AAcAAGUCUuAUCUUUGUGdTdTantis 1407 462 AcAAucuGGucuAAuuGuGdTdT sense 1108 463cAcAAUuAGACcAGAUUGUdTdT antis 1108 464 cAGucAuGcAcucAcAAAGdTdT sense1395 465 CUUUGUGAGUGcAUGACUGdTdT antis 1395 466 GAcAcAucGuucGAuuuAAdTdTsense 1595 467 UuAAAUCGAACGAUGUGUCdTdT antis 1595 468cuGcuAcccAGAAccuuuudTdT sense 1992 469 AAAAGGUUCUGGGuAGcAGdTdT antis1992 470 ucAGccAuAGcuuGAuuGcdTdT sense 1809 471 GcAAUcAAGCuAUGGCUGAdTdTantis 1809 472 AuuuAuuGccGuccuGGAcdTdT sense 1220 473GUCcAGGACGGcAAuAAAUdTdT antis 1220 474 cAAuuuGcAuAAuAcuAAudTdT sense1203 475 AUuAGuAUuAUGcAAAUUGdTdT antis 1203 476 GuAcAGucccAGcAcAuuudTdTsense 1322 477 AAAUGUGCUGGGACUGuACdTdT antis 1322 478uAccuucAGccAuAGcuuGdTdT sense 1804 479 cAAGCuAUGGCUGAAGGuAdTdT antis1804 480 AcAGAcAcuAcAuuAcccudTdT sense 1968 481 AGGGuAAUGuAGUGUCUGUdTdTantis 1968 482 AuAcuAAuuuAuuGccGucdTdT sense 1214 483GACGGcAAuAAAUuAGuAUdTdT antis 1214 484 GGGcuAuuGAAGAuAcAcAdTdT sense1159 485 UGUGuAUCUUcAAuAGCCCdTdT antis 1159 486 GuucGAuuuAAGccAucAudTdTsense 1603 487 AUGAUGGCUuAAAUCGAACdTdT antis 1603 488uGuGccuccuAGAcAcccGdTdT sense 1123 489 CGGGUGUCuAGGAGGcAcAdTdT antis1123 490 cuGGAcucuGuGuGAGcGudTdT sense 1233 491 ACGCUcAcAcAGAGUCcAGdTdTantis 1233 492 AcccucucuuucAAuuGcAdTdT sense 1930 493UGcAAUUGAAAGAGAGGGUdTdT antis 1930 494 cAGAcAcuAcAuuAcccuAdTdT sense1969 495 uAGGGuAAUGuAGUGUCUGdTdT antis 1969 496 AAuuuAuuGccGuccuGGAdTdTsense 1219 497 UCcAGGACGGcAAuAAAUUdTdT antis 1219 498uGuGuGAGcGuGuccAcAGdTdT sense 1241 499 CUGUGGAcACGCUcAcAcAdTdT antis1241 500 ccuGGuGGGcuAuuGAAGAdTdT sense 1153 501 UCUUcAAuAGCCcACcAGGdTdTantis 1153 502 AccuucAGccAuAGcuuGAdTdT sense 1805 503UcAAGCuAUGGCUGAAGGUdTdT antis 1805 504 GGAuGcuGAAGuAcAGuccdTdT sense1312 505 GGACUGuACUUcAGcAUCCdTdT antis 1312 506 AuccuAGuuccAuucuuGGdTdTsense 1546 507 CcAAGAAUGGAACuAGGAUdTdT antis 1546 508uccuAGuuccAuucuuGGudTdT sense 1547 509 ACcAAGAAUGGAACuAGGAdTdT antis1547 510 GGAGuAcAAucuGGucuAAdTdT sense 1103 511 UuAGACcAGAUUGuACUCCdTdTantis 1103 512 cAcAuuuccucucuAucuudTdT sense 1334 513AAGAuAGAGAGGAAAUGUGdTdT antis 1334 514 cAcAGAGuuuGuAGuAAAudTdT sense1255 515 AUUuACuAcAAACUCUGUGdTdT antis 1255 516 AAcAGAcAcuAcAuuAcccdTdTsense 1967 517 GGGuAAUGuAGUGUCUGUUdTdT antis 1967 518uucucAGucAuGcAcucAcdTdT sense 1391 519 GUGAGUGcAUGACUGAGAAdTdT antis1391 520 GuGccuccuAGAcAcccGcdTdT sense 1124 521 GCGGGUGUCuAGGAGGcACdTdTantis 1124 522 AAGccAucAucAGcuuAAudTdT sense 1612 523AUuAAGCUGAUGAUGGCUUdTdT antis 1612 524 cucucuuucAAuuGcAGAudTdT sense1933 525 AUCUGcAAUUGAAAGAGAGdTdT antis 1933 526 AcAccAuccuccAGuuGAAdTdTsense 1078 527 UUcAACUGGAGGAUGGUGUdTdT antis 1078 528uAuccuAGuuccAuucuuGdTdT sense 1545 529 cAAGAAUGGAACuAGGAuAdTdT antis1545 530 cAAucuGGucuAAuuGuGcdTdT sense 1109 531 GcAcAAUuAGACcAGAUUGdTdTantis 1109 532 ucAuGcAcucAcAAAGAuAdTdT sense 1398 533uAUCUUUGUGAGUGcAUGAdTdT antis 1398 534 AGAcAcuAcAuuAcccuAAdTdT sense1970 535 UuAGGGuAAUGuAGUGUCUdTdT antis 1970 536 AcAcAAGAGGGAcuGuAuudTdTsense 1173 537 AAuAcAGUCCCUCUUGUGUdTdT antis 1173 538GAuGcuGAAGuAcAGucccdTdT sense 1313 539 GGGACUGuACUUcAGcAUCdTdT antis1313 540 AGccAuAGcuuGAuuGcucdTdT sense 1811 541 GAGcAAUcAAGCuAUGGCUdTdTantis 1811 542 cAcAGGAGuccuuucuuuudTdT sense 1862 543AAAAGAAAGGACUCCUGUGdTdT antis 1862 544 AucGuucGAuuuAAGccAudTdT sense1600 545 AUGGCUuAAAUCGAACGAUdTdT antis 1600 546 ucAucAGcuuAAuuuAAGudTdTsense 1618 547 ACUuAAAUuAAGCUGAUGAdTdT antis 1618 548AGcAcAuuuccucucuAucdTdT sense 1332 549 GAuAGAGAGGAAAUGUGCUdTdT antis1332 550 GuGGGcuAuuGAAGAuAcAdTdT sense 1157 551 UGuAUCUUcAAuAGCCcACdTdTantis 1157 552 AucAuGuAuucccAucuAGdTdT sense 888 553CuAGAUGGGAAuAcAUGAUdTdT antis 888 554 AAAGAcAcAcAGGAGuccudTdT sense 1855555 AGGACUCCUGUGUGUCUUUdTdT antis 1855 556 cAAAuGAuAcAGucAGGAcdTdT sense1579 557 GUCCUGACUGuAUcAUUUGdTdT antis 1579 558 uuAGAAcAAuuAucAcAuAdTdTsense 805 559 uAUGUGAuAAUUGUUCuAAdTdT antis 805 560uccAuucuuGGucAAGuuudTdT sense 1554 561 AAACUUGACcAAGAAUGGAdTdT antis1554 562 cuGGucuAAuuGuGccuccdTdT sense 1113 563 GGAGGcAcAAUuAGACcAGdTdTantis 1113 564 cAcAAGAGGGAcuGuAuuudTdT sense 1174 565AAAuAcAGUCCCUCUUGUGdTdT antis 1174 566 ucuuGucucAcuuuGGAcudTdT sense1735 567 AGUCcAAAGUGAGAcAAGAdTdT antis 1735 568 uuuucuAuGGAGcAAAAcAdTdTsense 1450 569 UGUUUUGCUCcAuAGAAAAdTdT antis 1450 570AuuuAAAcuAuucAGAGGAdTdT sense 1285 571 UCCUCUGAAuAGUUuAAAUdTdT antis1285 572 uuuAGAAcAAuuAucAcAudTdT sense 804 573 AUGUGAuAAUUGUUCuAAAdTdTantis 804 574 GGAGuccuuucuuuuGAAAdTdT sense 1866 575UUUcAAAAGAAAGGACUCCdTdT antis 1866 576 uuAAGccAucAucAGcuuAdTdT sense1610 577 uAAGCUGAUGAUGGCUuAAdTdT antis 1610 578 ucuAAuuGuGccuccuAGAdTdTsense 1117 579 UCuAGGAGGcAcAAUuAGAdTdT antis 1117 580AAGuAcAGucccAGcAcAudTdT sense 1320 581 AUGUGCUGGGACUGuACUUdTdT antis1320 582 cuGAAGuAcAGucccAGcAdTdT sense 1317 583 UGCUGGGACUGuACUUcAGdTdTantis 1317 584 Modifications: Sense strand - all pyrimidines (U, C) are2′OMe; antisense strand - pyrimidines adjacent to A (UA, CA) are 2′Ome;3′end is thio (dTsdT). cuAAuuuAuuGccGuccuGdTsdT sense 1217 585cAGGACGGcAAuAAAUuAGdTsdT antis 1217 586 AAuAcuAAuuuAuuGccGudTsdT sense1213 587 ACGGcAAuAAAUuAGuAUUdTsdT antis 1213 588cAGccAuAGcuuGAuuGcudTsdT sense 1810 589 AGcAAUcAAGCuAUGGCUGdTsdT antis1810 590 GucAGGAcAcAucGuucGAdTsdT sense 1590 591UCGAACGAUGUGUCCUGACdTsdT antis 1590 592 cuucccuGGuGGGcuAuuGdTsdT sense1149 593 cAAuAGCCcACcAGGGAAGdTsdT antis 1149 594GAcAcuAcAuuAcccuAAudTsdT sense 1971 595 AUuAGGGuAAUGuAGUGUCdTsdT antis1971 596 AcucuGuGuGAGcGuGuccdTsdT sense 1237 597GGAcACGCUcAcAcAGAGUdTsdT antis 1237 598 cccuGGuGGGcuAuuGAAGdTsdT sense1152 599 CUUcAAuAGCCcACcAGGGdTsdT antis 1152 600AcuAAuuuAuuGccGuccudTsdT sense 1216 601 AGGACGGcAAuAAAUuAGUdTsdT antis1216 602 cucucAAAuGAuAcAGucAdTsdT sense 1575 603UGACUGuAUcAUUUGAGAGdTsdT antis 1575 604 AGuAcAAucuGGucuAAuudTsdT sense1105 605 AAUuAGACcAGAUUGuACUdTsdT antis 1105 606cAcAAAGAuAAGAcuuGuudTsdT sense 1407 607 AAcAAGUCUuAUCUUUGUGdTsdT antis1407 608 AcAAucuGGucuAAuuGuGdTsdT sense 1108 609cAcAAUuAGACcAGAUUGUdTsdT antis 1108 610 cAGucAuGcAcucAcAAAGdTsdT sense1395 611 CUUUGUGAGUGcAUGACUGdTsdT antis 1395 612GAcAcAucGuucGAuuuAAdTsdT sense 1595 613 UuAAAUCGAACGAUGUGUCdTsdT antis1595 614 cuGcuAcccAGAAccuuuudTsdT sense 1992 615AAAAGGUUCUGGGuAGcAGdTsdT antis 1992 616 ucAGccAuAGcuuGAuuGcdTsdT sense1809 617 GcAAUcAAGCuAUGGCUGAdTsdT antis 1809 618AuuuAuuGccGuccuGGAcdTsdT sense 1220 619 GUCcAGGACGGcAAuAAAUdTsdT antis1220 620 cAAuuuGcAuAAuAcuAAudTsdT sense 1203 621AUuAGuAUuAUGcAAAUUGdTsdT antis 1203 622 GuAcAGucccAGcAcAuuudTsdT sense1322 623 AAAUGUGCUGGGACUGuACdTsdT antis 1322 624uAccuucAGccAuAGcuuGdTsdT sense 1804 625 cAAGCuAUGGCUGAAGGuAdTsdT antis1804 626 AcAGAcAcuAcAuuAcccudTsdT sense 1968 627AGGGuAAUGuAGUGUCUGUdTsdT antis 1968 628 AuAcuAAuuuAuuGccGucdTsdT sense1214 629 GACGGcAAuAAAUuAGuAUdTsdT antis 1214 630GGGcuAuuGAAGAuAcAcAdTsdT sense 1159 631 UGUGuAUCUUcAAuAGCCCdTsdT antis1159 632 GuucGAuuuAAGccAucAudTsdT sense 1603 633AUGAUGGCUuAAAUCGAACdTsdT antis 1603 634 uGuGccuccuAGAcAcccGdTsdT sense1123 635 CGGGUGUCuAGGAGGcAcAdTsdT antis 1123 636cuGGAcucuGuGuGAGcGudTsdT sense 1233 637 ACGCUcAcAcAGAGUCcAGdTsdT antis1233 638 AcccucucuuucAAuuGcAdTsdT sense 1930 639UGcAAUUGAAAGAGAGGGUdTsdT antis 1930 640 cAGAcAcuAcAuuAcccuAdTsdT sense1969 641 uAGGGuAAUGuAGUGUCUGdTsdT antis 1969 642AAuuuAuuGccGuccuGGAdTsdT sense 1219 643 UCcAGGACGGcAAuAAAUUdTsdT antis1219 644 uGuGuGAGcGuGuccAcAGdTsdT sense 1241 645CUGUGGAcACGCUcAcAcAdTsdT antis 1241 646 ccuGGuGGGcuAuuGAAGAdTsdT sense1153 647 UCUUcAAuAGCCcACcAGGdTsdT antis 1153 648AccuucAGccAuAGcuuGAdTsdT sense 1805 649 UcAAGCuAUGGCUGAAGGUdTsdT antis1805 650 GGAuGcuGAAGuAcAGuccdTsdT sense 1312 651GGACUGuACUUcAGcAUCCdTsdT antis 1312 652 AuccuAGuuccAuucuuGGdTsdT sense1546 653 CcAAGAAUGGAACuAGGAUdTsdT antis 1546 654uccuAGuuccAuucuuGGudTsdT sense 1547 655 ACcAAGAAUGGAACuAGGAdTsdT antis1547 656 GGAGuAcAAucuGGucuAAdTsdT sense 1103 657UuAGACcAGAUUGuACUCCdTsdT antis 1103 658 cAcAuuuccucucuAucuudTsdT sense1334 659 AAGAuAGAGAGGAAAUGUGdTsdT antis 1334 660cAcAGAGuuuGuAGuAAAudTsdT sense 1255 661 AUUuACuAcAAACUCUGUGdTsdT antis1255 662 AAcAGAcAcuAcAuuAcccdTsdT sense 1967 663GGGuAAUGuAGUGUCUGUUdTsdT antis 1967 664 uucucAGucAuGcAcucAcdTsdT sense1391 665 GUGAGUGcAUGACUGAGAAdTsdT antis 1391 666GuGccuccuAGAcAcccGcdTsdT sense 1124 667 GCGGGUGUCuAGGAGGcACdTsdT antis1124 668 AAGccAucAucAGcuuAAudTsdT sense 1612 669AUuAAGCUGAUGAUGGCUUdTsdT antis 1612 670 cucucuuucAAuuGcAGAudTsdT sense1933 671 AUCUGcAAUUGAAAGAGAGdTsdT antis 1933 672AcAccAuccuccAGuuGAAdTsdT sense 1078 673 UUcAACUGGAGGAUGGUGUdTsdT antis1078 674 uAuccuAGuuccAuucuuGdTsdT sense 1545 675cAAGAAUGGAACuAGGAuAdTsdT antis 1545 676 cAAucuGGucuAAuuGuGcdTsdT sense1109 677 GcAcAAUuAGACcAGAUUGdTsdT antis 1109 678ucAuGcAcucAcAAAGAuAdTsdT sense 1398 679 uAUCUUUGUGAGUGcAUGAdTsdT antis1398 680 AGAcAcuAcAuuAcccuAAdTsdT sense 1970 681UuAGGGuAAUGuAGUGUCUdTsdT antis 1970 682 AcAcAAGAGGGAcuGuAuudTsdT sense1173 683 AAuAcAGUCCCUCUUGUGUdTsdT antis 1173 684GAuGcuGAAGuAcAGucccdTsdT sense 1313 685 GGGACUGuACUUcAGcAUCdTsdT antis1313 686 AGccAuAGcuuGAuuGcucdTsdT sense 1811 687GAGcAAUcAAGCuAUGGCUdTsdT antis 1811 688 cAcAGGAGuccuuucuuuudTsdT sense1862 689 AAAAGAAAGGACUCCUGUGdTsdT antis 1862 690AucGuucGAuuuAAGccAudTsdT sense 1600 691 AUGGCUuAAAUCGAACGAUdTsdT antis1600 692 ucAucAGcuuAAuuuAAGudTsdT sense 1618 693ACUuAAAUuAAGCUGAUGAdTsdT antis 1618 694 AGcAcAuuuccucucuAucdTsdT sense1332 695 GAuAGAGAGGAAAUGUGCUdTsdT antis 1332 696GuGGGcuAuuGAAGAuAcAdTsdT sense 1157 697 UGuAUCUUcAAuAGCCcACdTsdT antis1157 698 AucAuGuAuucccAucuAGdTsdT sense 888 699 CuAGAUGGGAAuAcAUGAUdTsdTantis 888 700 AAAGAcAcAcAGGAGuccudTsdT sense 1855 701AGGACUCCUGUGUGUCUUUdTsdT antis 1855 702 cAAAuGAuAcAGucAGGAcdTsdT sense1579 703 GUCCUGACUGuAUcAUUUGdTsdT antis 1579 704uuAGAAcAAuuAucAcAuAdTsdT sense 805 705 uAUGUGAuAAUUGUUCuAAdTsdT antis805 706 uccAuucuuGGucAAGuuudTsdT sense 1554 707 AAACUUGACcAAGAAUGGAdTsdTantis 1554 708 cuGGucuAAuuGuGccuccdTsdT sense 1113 709GGAGGcAcAAUuAGACcAGdTsdT antis 1113 710 cAcAAGAGGGAcuGuAuuudTsdT sense1174 711 AAAuAcAGUCCCUCUUGUGdTsdT antis 1174 712ucuuGucucAcuuuGGAcudTsdT sense 1735 713 AGUCcAAAGUGAGAcAAGAdTsdT antis1735 714 uuuucuAuGGAGcAAAAcAdTsdT sense 1450 715UGUUUUGCUCcAuAGAAAAdTsdT antis 1450 716 AuuuAAAcuAuucAGAGGAdTsdT sense1285 717 UCCUCUGAAuAGUUuAAAUdTsdT antis 1285 718uuuAGAAcAAuuAucAcAudTsdT sense 804 719 AUGUGAuAAUUGUUCuAAAdTsdT antis804 720 GGAGuccuuucuuuuGAAAdTsdT sense 1866 721 UUUcAAAAGAAAGGACUCCdTsdTantis 1866 722 uuAAGccAucAucAGcuuAdTsdT sense 1610 723uAAGCUGAUGAUGGCUuAAdTsdT antis 1610 724 ucuAAuuGuGccuccuAGAdTsdT sense1117 725 UCuAGGAGGcAcAAUuAGAdTsdT antis 1117 726AAGuAcAGucccAGcAcAudTsdT sense 1320 727 AUGUGCUGGGACUGuACUUdTsdT antis1320 728 cuGAAGuAcAGucccAGcAdTsdT sense 1317 729UGCUGGGACUGuACUUcAGdTsdT antis 1317 730 Modifications: Sense strand -all pyrimidines are 2′OMe; antisense strand - pyrimidines adjacent to A(UA, CA) + U adjacent to another U (UU) or G (UG) are 2′Ome; 3′ end isthio (dTsdT). cuAAuuuAuuGccGuccuGdTsdT sense 1217 731cAGGACGGcAAuAAAUuAGdTsdT antis 1217 732 AAuAcuAAuuuAuuGccGudTsdT sense1213 733 ACGGcAAuAAAUuAGuAuUdTsdT antis 1213 734cAGccAuAGcuuGAuuGcudTsdT sense 1810 735 AGcAAUcAAGCuAuGGCuGdTsdT antis1810 736 GucAGGAcAcAucGuucGAdTsdT sense 1590 737UCGAACGAuGuGUCCuGACdTsdT antis 1590 738 cuucccuGGuGGGcuAuuGdTsdT sense1149 739 cAAuAGCCcACcAGGGAAGdTsdT antis 1149 740GAcAcuAcAuuAcccuAAudTsdT sense 1971 741 AUuAGGGuAAuGuAGuGUCdTsdT antis1971 742 AcucuGuGuGAGcGuGuccdTsdT sense 1237 743GGAcACGCUcAcAcAGAGUdTsdT antis 1237 744 cccuGGuGGGcuAuuGAAGdTsdT sense1152 745 CuUcAAuAGCCcACcAGGGdTsdT antis 1152 746AcuAAuuuAuuGccGuccudTsdT sense 1216 747 AGGACGGcAAuAAAUuAGUdTsdT antis1216 748 cucucAAAuGAuAcAGucAdTsdT sense 1575 749uGACuGuAUcAuUuGAGAGdTsdT antis 1575 750 AGuAcAAucuGGucuAAuudTsdT sense1105 751 AAUuAGACcAGAuuGuACUdTsdT antis 1105 752cAcAAAGAuAAGAcuuGuudTsdT sense 1407 753 AAcAAGUCUuAUCuUuGuGdTsdT antis1407 754 AcAAucuGGucuAAuuGuGdTsdT sense 1108 755cAcAAUuAGACcAGAuuGUdTsdT antis 1108 756 cAGucAuGcAcucAcAAAGdTsdT sense1395 757 CuUuGuGAGuGcAuGACuGdTsdT antis 1395 758GAcAcAucGuucGAuuuAAdTsdT sense 1595 759 UuAAAUCGAACGAuGuGUCdTsdT antis1595 760 cuGcuAcccAGAAccuuuudTsdT sense 1992 761AAAAGGuUCuGGGuAGcAGdTsdT antis 1992 762 ucAGccAuAGcuuGAuuGcdTsdT sense1809 763 GcAAUcAAGCuAuGGCuGAdTsdT antis 1809 764AuuuAuuGccGuccuGGAcdTsdT sense 1220 765 GUCcAGGACGGcAAuAAAUdTsdT antis1220 766 cAAuuuGcAuAAuAcuAAudTsdT sense 1203 767AUuAGuAUuAuGcAAAuuGdTsdT antis 1203 768 GuAcAGucccAGcAcAuuudTsdT sense1322 769 AAAuGuGCuGGGACuGuACdTsdT antis 1322 770uAccuucAGccAuAGcuuGdTsdT sense 1804 771 cAAGCuAuGGCuGAAGGuAdTsdT antis1804 772 AcAGAcAcuAcAuuAcccudTsdT sense 1968 773AGGGuAAuGuAGuGUCuGUdTsdT antis 1968 774 AuAcuAAuuuAuuGccGucdTsdT sense1214 775 GACGGcAAuAAAUuAGuAUdTsdT antis 1214 776GGGcuAuuGAAGAuAcAcAdTsdT sense 1159 777 uGuGuAUCuUcAAuAGCCCdTsdT antis1159 778 GuucGAuuuAAGccAucAudTsdT sense 1603 779AuGAuGGCUuAAAUCGAACdTsdT antis 1603 780 uGuGccuccuAGAcAcccGdTsdT sense1123 781 CGGGuGUCuAGGAGGcAcAdTsdT antis 1123 782cuGGAcucuGuGuGAGcGudTsdT sense 1233 783 ACGCUcAcAcAGAGUCcAGdTsdT antis1233 784 AcccucucuuucAAuuGcAdTsdT sense 1930 785uGcAAuuGAAAGAGAGGGUdTsdT antis 1930 786 cAGAcAcuAcAuuAcccuAdTsdT sense1969 787 uAGGGuAAuGuAGuGUCuGdTsdT antis 1969 788AAuuuAuuGccGuccuGGAdTsdT sense 1219 789 UCcAGGACGGcAAuAAAuUdTsdT antis1219 790 uGuGuGAGcGuGuccAcAGdTsdT sense 1241 791CuGuGGAcACGCUcAcAcAdTsdT antis 1241 792 ccuGGuGGGcuAuuGAAGAdTsdT sense1153 793 UCuUcAAuAGCCcACcAGGdTsdT antis 1153 794AccuucAGccAuAGcuuGAdTsdT sense 1805 795 UcAAGCuAuGGCuGAAGGUdTsdT antis1805 796 GGAuGcuGAAGuAcAGuccdTsdT sense 1312 797GGACuGuACuUcAGcAUCCdTsdT antis 1312 798 AuccuAGuuccAuucuuGGdTsdT sense1546 799 CcAAGAAuGGAACuAGGAUdTsdT antis 1546 800uccuAGuuccAuucuuGGudTsdT sense 1547 801 ACcAAGAAuGGAACuAGGAdTsdT antis1547 802 GGAGuAcAAucuGGucuAAdTsdT sense 1103 803UuAGACcAGAuuGuACUCCdTsdT antis 1103 804 cAcAuuuccucucuAucuudTsdT sense1334 805 AAGAuAGAGAGGAAAuGuGdTsdT antis 1334 806cAcAGAGuuuGuAGuAAAudTsdT sense 1255 807 AuUuACuAcAAACUCuGuGdTsdT antis1255 808 AAcAGAcAcuAcAuuAcccdTsdT sense 1967 809GGGuAAuGuAGuGUCuGuUdTsdT antis 1967 810 uucucAGucAuGcAcucAcdTsdT sense1391 811 GuGAGuGcAuGACuGAGAAdTsdT antis 1391 812GuGccuccuAGAcAcccGcdTsdT sense 1124 813 GCGGGuGUCuAGGAGGcACdTsdT antis1124 814 AAGccAucAucAGcuuAAudTsdT sense 1612 815AUuAAGCuGAuGAuGGCuUdTsdT antis 1612 816 cucucuuucAAuuGcAGAudTsdT sense1933 817 AUCuGcAAuuGAAAGAGAGdTsdT antis 1933 818AcAccAuccuccAGuuGAAdTsdT sense 1078 819 uUcAACuGGAGGAuGGuGUdTsdT antis1078 820 uAuccuAGuuccAuucuuGdTsdT sense 1545 821cAAGAAuGGAACuAGGAuAdTsdT antis 1545 822 cAAucuGGucuAAuuGuGcdTsdT sense1109 823 GcAcAAUuAGACcAGAuuGdTsdT antis 1109 824ucAuGcAcucAcAAAGAuAdTsdT sense 1398 825 uAUCuUuGuGAGuGcAuGAdTsdT antis1398 826 AGAcAcuAcAuuAcccuAAdTsdT sense 1970 827UuAGGGuAAuGuAGuGUCUdTsdT antis 1970 828 AcAcAAGAGGGAcuGuAuudTsdT sense1173 829 AAuAcAGUCCCUCuuGuGUdTsdT antis 1173 830GAuGcuGAAGuAcAGucccdTsdT sense 1313 831 GGGACuGuACuUcAGcAUCdTsdT antis1313 832 AGccAuAGcuuGAuuGcucdTsdT sense 1811 833GAGcAAUcAAGCuAuGGCUdTsdT antis 1811 834 cAcAGGAGuccuuucuuuudTsdT sense1862 835 AAAAGAAAGGACUCCuGuGdTsdT antis 1862 836AucGuucGAuuuAAGccAudTsdT sense 1600 837 AuGGCUuAAAUCGAACGAUdTsdT antis1600 838 ucAucAGcuuAAuuuAAGudTsdT sense 1618 839ACUuAAAUuAAGCuGAuGAdTsdT antis 1618 840 AGcAcAuuuccucucuAucdTsdT sense1332 841 GAuAGAGAGGAAAuGuGCUdTsdT antis 1332 842GuGGGcuAuuGAAGAuAcAdTsdT sense 1157 843 uGuAUCuUcAAuAGCCcACdTsdT antis1157 844 AucAuGuAuucccAucuAGdTsdT sense 888 845 CuAGAuGGGAAuAcAuGAUdTsdTantis 888 846 AAAGAcAcAcAGGAGuccudTsdT sense 1855 847AGGACUCCuGuGuGUCuUUdTsdT antis 1855 848 cAAAuGAuAcAGucAGGAcdTsdT sense1579 849 GUCCuGACuGuAUcAuUuGdTsdT antis 1579 850uuAGAAcAAuuAucAcAuAdTsdT sense 805 851 uAuGuGAuAAuuGuUCuAAdTsdT antis805 852 uccAuucuuGGucAAGuuudTsdT sense 1554 853 AAACuuGACcAAGAAuGGAdTsdTantis 1554 854 cuGGucuAAuuGuGccuccdTsdT sense 1113 855GGAGGcAcAAUuAGACcAGdTsdT antis 1113 856 cAcAAGAGGGAcuGuAuuudTsdT sense1174 857 AAAuAcAGUCCCUCuuGuGdTsdT antis 1174 858ucuuGucucAcuuuGGAcudTsdT sense 1735 859 AGUCcAAAGuGAGAcAAGAdTsdT antis1735 860 uuuucuAuGGAGcAAAAcAdTsdT sense 1450 861uGuUuuGCUCcAuAGAAAAdTsdT antis 1450 862 AuuuAAAcuAuucAGAGGAdTsdT sense1285 863 UCCUCuGAAuAGuUuAAAUdTsdT antis 1285 864uuuAGAAcAAuuAucAcAudTsdT sense 804 865 AuGuGAuAAuuGuUCuAAAdTsdT antis804 866 GGAGuccuuucuuuuGAAAdTsdT sense 1866 867 uUUcAAAAGAAAGGACUCCdTsdTantis 1866 868 uuAAGccAucAucAGcuuAdTsdT sense 1610 869uAAGCuGAuGAuGGCUuAAdTsdT antis 1610 870 ucuAAuuGuGccuccuAGAdTsdT sense1117 871 UCuAGGAGGcAcAAUuAGAdTsdT antis 1117 872AAGuAcAGucccAGcAcAudTsdT sense 1320 873 AuGuGCuGGGACuGuACuUdTsdT antis1320 874 cuGAAGuAcAGucccAGcAdTsdT sense 1317 875uGCuGGGACuGuACuUcAGdTsdT antis 1317 876

TABLE 3a GNAQ (Human, monkey and mouse): target sequencesNumbering for target sequence is Human GNAQ NM_002072. Start of SEQ SEQtarget ID Target sequence, ID Target sequence, sequence NO.sense strand (5′-3′) NO. antisense strand (5′-3′) 1215 877UACUAAUUUAUUGCCGUCC 888 GGACGGCAAUAAAUUAGUA 1217 878 CUAAUUUAUUGCCGUCCUG889 CAGGACGGCAAUAAAUUAG 1216 879 ACUAAUUUAUUGCCGUCCU 890AGGACGGCAAUAAAUUAGU 1322 880 GUACAGUCCCAGCACAUUU 891 AAAUGUGCUGGGACUGUAC1220 881 AUUUAUUGCCGUCCUGGAC 892 GUCCAGGACGGCAAUAAAU 1265 882GUAGUAAAUAUUAUGAUUU 893 AAAUCAUAAUAUUUACUAC 1218 883 UAAUUUAUUGCCGUCCUGG894 CCAGGACGGCAAUAAAUUA 1175 884 ACAAGAGGGACUGUAUUUC 895GAAAUACAGUCCCUCUUGU 1223 885 UAUUGCCGUCCUGGACUCU 896 AGAGUCCAGGACGGCAAUA1319 886 GAAGUACAGUCCCAGCACA 897 UGUGCUGGGACUGUACUUC 1285 887AUUUAAACUAUUCAGAGGA 898 UCCUCUGAAUAGUUUAAAU

TABLE 3b GNAQ (Human, monkey and mouse): sense andantisense sequences with 2 base overhangsNumbering for target sequence is Human GNAQ NM_002072. SEQ Start of IDtarget NO SEQUENCE (5′-3′) Strand sequence 899 UACUAAUUUAUUGCCGUCCNNsense 1215 900 GGACGGCAAUAAAUUAGUANN antis 1215 901CUAAUUUAUUGCCGUCCUGNN sense 1217 902 CAGGACGGCAAUAAAUUAGNN antis 1217903 ACUAAUUUAUUGCCGUCCUNN sense 1216 904 AGGACGGCAAUAAAUUAGUNN antis1216 905 GUACAGUCCCAGCACAUUUNN sense 1322 906 AAAUGUGCUGGGACUGUACNNantis 1322 907 AUUUAUUGCCGUCCUGGACNN sense 1220 908GUCCAGGACGGCAAUAAAUNN antis 1220 909 GUAGUAAAUAUUAUGAUUUNN sense 1265910 AAAUCAUAAUAUUUACUACNN antis 1265 911 UAAUUUAUUGCCGUCCUGGNN sense1218 912 CCAGGACGGCAAUAAAUUANN antis 1218 913 ACAAGAGGGACUGUAUUUCNNsense 1175 914 GAAAUACAGUCCCUCUUGUNN antis 1175 915UAUUGCCGUCCUGGACUCUNN sense 1223 916 AGAGUCCAGGACGGCAAUANN antis 1223917 GAAGUACAGUCCCAGCACANN sense 1319 918 UGUGCUGGGACUGUACUUCNN antis1319 919 AUUUAAACUAUUCAGAGGANN sense 1285 920 UCCUCUGAAUAGUUUAAAUNNantis 1285

TABLE 3c GNAQ (Human, monkey and mouse): sense andantisense sequences with dTdT overhangsNumbering for target sequence is Human GNAQ NM_002072. SEQ Start of IDtarget NO SEQUENCE (5′-3′) Strand sequence 921 UACUAAUUUAUUGCCGUCCdTdTsense 1215 922 GGACGGCAAUAAAUUAGUAdTdT antis 1215 923CUAAUUUAUUGCCGUCCUGdTdT sense 1217 924 CAGGACGGCAAUAAAUUAGdTdT antis1217 925 ACUAAUUUAUUGCCGUCCUdTdT sense 1216 926 AGGACGGCAAUAAAUUAGUdTdTantis 1216 927 GUACAGUCCCAGCACAUUUdTdT sense 1322 928AAAUGUGCUGGGACUGUACdTdT antis 1322 929 AUUUAUUGCCGUCCUGGACdTdT sense1220 930 GUCCAGGACGGCAAUAAAUdTdT antis 1220 931 GUAGUAAAUAUUAUGAUUUdTdTsense 1265 932 AAAUCAUAAUAUUUACUACdTdT antis 1265 933UAAUUUAUUGCCGUCCUGGdTdT sense 1218 934 CCAGGACGGCAAUAAAUUAdTdT antis1218 935 ACAAGAGGGACUGUAUUUCdTdT sense 1175 936 GAAAUACAGUCCCUCUUGUdTdTantis 1175 937 UAUUGCCGUCCUGGACUCUdTdT sense 1223 938AGAGUCCAGGACGGCAAUAdTdT antis 1223 939 GAAGUACAGUCCCAGCACAdTdT sense1319 940 UGUGCUGGGACUGUACUUCdTdT antis 1319 941 AUUUAAACUAUUCAGAGGAdTdTsense 1285 942 UCCUCUGAAUAGUUUAAAUdTdT antis 1285

TABLE 3d GNAQ (Human, monkey and mouse): modified senseandantisense strands Numbering for target sequence is Human GNAQNM_002072. Start of target SEQ SEQUENCE (5′-3′) Strand sequence ID NOModifications: Sense strand - all pyrimidines(U, C) are 2′OMe; antisense strand - pyrimidinesadjacent to A (UA, CA) are 2′Ome; 3′ end is dTdT uAcuAAuuuAuuGccGuccdTdTsense 1215 943 GGACGGcAAuAAAUuAGuAdTdT antis 1215 944cuAAuuuAuuGccGuccuGdTdT sense 1217 945 cAGGACGGcAAuAAAUuAGdTdT antis1217 946 AcuAAuuuAuuGccGuccudTdT sense 1216 947 AGGACGGcAAuAAAUuAGUdTdTantis 1216 948 GuAcAGucccAGcAcAuuudTdT sense 1322 949AAAUGUGCUGGGACUGuACdTdT antis 1322 950 AuuuAuuGccGuccuGGAcdTdT sense1220 951 GUCcAGGACGGcAAuAAAUdTdT antis 1220 952 GuAGuAAAuAuuAuGAuuudTdTsense 1265 953 AAAUcAuAAuAUUuACuACdTdT antis 1265 954uAAuuuAuuGccGuccuGGdTdT sense 1218 955 CcAGGACGGcAAuAAAUuAdTdT antis1218 956 AcAAGAGGGAcuGuAuuucdTdT sense 1175 957 GAAAuAcAGUCCCUCUUGUdTdTantis 1175 958 uAuuGccGuccuGGAcucudTdT sense 1223 959AGAGUCcAGGACGGcAAuAdTdT antis 1223 960 GAAGuAcAGucccAGcAcAdTdT sense1319 961 UGUGCUGGGACUGuACUUCdTdT antis 1319 962 AuuuAAAcuAuucAGAGGAdTdTsense 1285 963 UCCUCUGAAuAGUUuAAAUdTdT antis 1285 964Modifications: Sense strand - all pyrimidines(U, C) are 2′OMe; antisense strand - pyrimidinesadjacent to A (UA, CA) are 2′Ome; 3′ end is thio (dTsdT)uAcuAAuuuAuuGccGuccdTsdT sense 1215 965 GGACGGcAAuAAAUuAGuAdTsdT antis1215 966 cuAAuuuAuuGccGuccuGdTsdT sense 1217 967cAGGACGGcAAuAAAUuAGdTsdT antis 1217 968 AcuAAuuuAuuGccGuccudTsdT sense1216 969 AGGACGGcAAuAAAUuAGUdTsdT antis 1216 970GuAcAGucccAGcAcAuuudTsdT sense 1322 971 AAAUGUGCUGGGACUGuACdTsdT antis1322 972 AuuuAuuGccGuccuGGAcdTsdT sense 1220 973GUCcAGGACGGcAAuAAAUdTsdT antis 1220 974 GuAGuAAAuAuuAuGAuuudTsdT sense1265 975 AAAUcAuAAuAUUuACuACdTsdT antis 1265 976uAAuuuAuuGccGuccuGGdTsdT sense 1218 977 CcAGGACGGcAAuAAAUuAdTsdT antis1218 978 AcAAGAGGGAcuGuAuuucdTsdT sense 1175 979GAAAuAcAGUCCCUCUUGUdTsdT antis 1175 980 uAuuGccGuccuGGAcucudTsdT sense1223 981 AGAGUCcAGGACGGcAAuAdTsdT antis 1223 982GAAGuAcAGucccAGcAcAdTsdT sense 1319 983 UGUGCUGGGACUGuACUUCdTsdT antis1319 984 AuuuAAAcuAuucAGAGGAdTsdT sense 1285 985UCCUCUGAAuAGUUuAAAUdTsdT antis 1285 986Modifications: Sense strand - all pyrimidines are2′OMe; antisense strand - pyrimidines adjacent to A (UA, CA) +U adjacent to another U (UU) are 2′Ome; 3′ end is thio (dTsdT).uAcuAAuuuAuuGccGuccdTsdT sense 1215 987 GGACGGcAAuAAAUuAGuAdTsdT antis1215 988 cuAAuuuAuuGccGuccuGdTsdT sense 1217 989cAGGACGGcAAuAAAUuAGdTsdT antis 1217 990 AcuAAuuuAuuGccGuccudTsdT sense1216 991 AGGACGGcAAuAAAUuAGUdTsdT antis 1216 992GuAcAGucccAGcAcAuuudTsdT sense 1322 993 AAAuGuGCuGGGACuGuACdTsdT antis1322 994 AuuuAuuGccGuccuGGAcdTsdT sense 1220 995GUCcAGGACGGcAAuAAAUdTsdT antis 1220 996 GuAGuAAAuAuuAuGAuuudTsdT sense1265 997 AAAUcAuAAuAuUuACuACdTsdT antis 1265 998uAAuuuAuuGccGuccuGGdTsdT sense 1218 999 CcAGGACGGcAAuAAAUuAdTsdT antis1218 1000 AcAAGAGGGAcuGuAuuucdTsdT sense 1175 1001GAAAuAcAGUCCCUCuuGUdTsdT antis 1175 1002 uAuuGccGuccuGGAcucudTsdT sense1223 1003 AGAGUCcAGGACGGcAAuAdTsdT antis 1223 1004GAAGuAcAGucccAGcAcAdTsdT sense 1319 1005 uGuGCuGGGACuGuACuUCdTsdT antis1319 1006 AuuuAAAcuAuucAGAGGAdTsdT sense 1285 1007UCCUCuGAAuAGuUuAAAUdTsdT antis 1285 1008

TABLE 4a GNAQ (rat and mouse): target sequencesNumbering for target sequences is Rat GNAQ NM_031036. Start of SEQTarget sequence, target SEQ ID Target sequence, ID antisense strandsequence NO. sense strand (5′-3′) NO. (5′-3′) 853 1009UAUUCCCACCUAGUCGACU 1039 AGUCGACUAGGUGGGAAUA 855 1010UUCCCACCUAGUCGACUAC 1040 GUAGUCGACUAGGUGGGAA 367 1011GCUUUUGAGAAUCCAUAUG 1041 CAUAUGGAUUCUCAAAAGC 55 1012 CGGAGGAUCAACGACGAGA1042 UCUCGUCGUUGAUCCUCCG 459 1013 AUCUGACUCUACCAAAUAC 1043GUAUUUGGUAGAGUCAGAU 312 1014 ACACAAUAAGGCUCAUGCA 1044UGCAUGAGCCUUAUUGUGU 178 1015 AGGAUCAUCCACGGGUCGG 1045CCGACCCGUGGAUGAUCCU 297 1016 CCCAUACAAGUAUGAACAC 1046GUGUUCAUACUUGUAUGGG 315 1017 CAAUAAGGCUCAUGCACAA 1047UUGUGCAUGAGCCUUAUUG 58 1018 AGGAUCAACGACGAGAUCG 1048 CGAUCUCGUCGUUGAUCCU324 1019 UCAUGCACAAUUGGUUCGA 1049 UCGAACCAAUUGUGCAUGA 59 1020GGAUCAACGACGAGAUCGA 1050 UCGAUCUCGUCGUUGAUCC 398 1021AGAGCUUGUGGAAUGAUCC 1051 GGAUCAUUCCACAAGCUCU 57 1022 GAGGAUCAACGACGAGAUC1052 GAUCUCGUCGUUGAUCCUC 56 1023 GGAGGAUCAACGACGAGAU 1053AUCUCGUCGUUGAUCCUCC 369 1024 UUUUGAGAAUCCAUAUGUA 1054UACAUAUGGAUUCUCAAAA 45 1025 CAAGGAAGCCCGGAGGAUC 1055 GAUCCUCCGGGCUUCCUUG460 1026 UCUGACUCUACCAAAUACU 1056 AGUAUUUGGUAGAGUCAGA 97 1027AAGCGCGACGCCCGCCGGG 1057 CCCGGCGGGCGUCGCGCUU 314 1028ACAAUAAGGCUCAUGCACA 1058 UGUGCAUGAGCCUUAUUGU 318 1029UAAGGCUCAUGCACAAUUG 1059 CAAUUGUGCAUGAGCCUUA 50 1030 AAGCCCGGAGGAUCAACGA1060 UCGUUGAUCCUCCGGGCUU 323 1031 CUCAUGCACAAUUGGUUCG 1061CGAACCAAUUGUGCAUGAG 327 1032 UGCACAAUUGGUUCGAGAG 1062CUCUCGAACCAAUUGUGCA 329 1033 CACAAUUGGUUCGAGAGGU 1063ACCUCUCGAACCAAUUGUG 862 1034 CUAGUCGACUACUUCCCAG 1064CUGGGAAGUAGUCGACUAG 89 1035 GCAGGGACAAGCGCGACGC 1065 GCGUCGCGCUUGUCCCUGC371 1036 UUGAGAAUCCAUAUGUAGA 1066 UCUACAUAUGGAUUCUCAA 868 1037GACUACUUCCCAGAAUAUG 1067 CAUAUUCUGGGAAGUAGUC 62 1038 UCAACGACGAGAUCGAGCG1068 CGCUCGAUCUCGUCGUUGA

TABLE 4b GNAQ (rat and mouse): sense and antisensesequences with 2 base overhangsNumbering for target sequences is Rat GNAQ NM_031036. SEQ IDStart of target NO SEQUENCE (5′-3′) Type sequence 1069UAUUCCCACCUAGUCGACUNN sense 853 1070 AGUCGACUAGGUGGGAAUANN antis 8531071 UUCCCACCUAGUCGACUACNN sense 855 1072 GUAGUCGACUAGGUGGGAANN antis855 1073 GCUUUUGAGAAUCCAUAUGNN sense 367 1074 CAUAUGGAUUCUCAAAAGCNNantis 367 1075 CGGAGGAUCAACGACGAGANN sense 55 1076 UCUCGUCGUUGAUCCUCCGNNantis 55 1077 AUCUGACUCUACCAAAUACNN sense 459 1078 GUAUUUGGUAGAGUCAGAUNNantis 459 1079 ACACAAUAAGGCUCAUGCANN sense 312 1080UGCAUGAGCCUUAUUGUGUNN antis 312 1081 AGGAUCAUCCACGGGUCGGNN sense 1781082 CCGACCCGUGGAUGAUCCUNN antis 178 1083 CCCAUACAAGUAUGAACACNN sense297 1084 GUGUUCAUACUUGUAUGGGNN antis 297 1085 CAAUAAGGCUCAUGCACAANNsense 315 1086 UUGUGCAUGAGCCUUAUUGNN antis 315 1087AGGAUCAACGACGAGAUCGNN sense 58 1088 CGAUCUCGUCGUUGAUCCUNN antis 58 1089UCAUGCACAAUUGGUUCGANN sense 324 1090 UCGAACCAAUUGUGCAUGANN antis 3241091 GGAUCAACGACGAGAUCGANN sense 59 1092 UCGAUCUCGUCGUUGAUCCNN antis 591093 AGAGCUUGUGGAAUGAUCCNN sense 398 1094 GGAUCAUUCCACAAGCUCUNN antis398 1095 GAGGAUCAACGACGAGAUCNN sense 57 1096 GAUCUCGUCGUUGAUCCUCNN antis57 1097 GGAGGAUCAACGACGAGAUNN sense 56 1098 AUCUCGUCGUUGAUCCUCCNN antis56 1099 UUUUGAGAAUCCAUAUGUANN sense 369 1100 UACAUAUGGAUUCUCAAAANN antis369 1101 CAAGGAAGCCCGGAGGAUCNN sense 45 1102 GAUCCUCCGGGCUUCCUUGNN antis45 1103 UCUGACUCUACCAAAUACUNN sense 460 1104 AGUAUUUGGUAGAGUCAGANN antis460 1105 AAGCGCGACGCCCGCCGGGNN sense 97 1106 CCCGGCGGGCGUCGCGCUUNN antis97 1107 ACAAUAAGGCUCAUGCACANN sense 314 1108 UGUGCAUGAGCCUUAUUGUNN antis314 1109 UAAGGCUCAUGCACAAUUGNN sense 318 1110 CAAUUGUGCAUGAGCCUUANNantis 318 1111 AAGCCCGGAGGAUCAACGANN sense 50 1112 UCGUUGAUCCUCCGGGCUUNNantis 50 1113 CUCAUGCACAAUUGGUUCGNN sense 323 1114 CGAACCAAUUGUGCAUGAGNNantis 323 1115 UGCACAAUUGGUUCGAGAGNN sense 327 1116CUCUCGAACCAAUUGUGCANN antis 327 1117 CACAAUUGGUUCGAGAGGUNN sense 3291118 ACCUCUCGAACCAAUUGUGNN antis 329 1119 CUAGUCGACUACUUCCCAGNN sense862 1120 CUGGGAAGUAGUCGACUAGNN antis 862 1121 GCAGGGACAAGCGCGACGCNNsense 89 1122 GCGUCGCGCUUGUCCCUGCNN antis 89 1123 UUGAGAAUCCAUAUGUAGANNsense 371 1124 UCUACAUAUGGAUUCUCAANN antis 371 1125GACUACUUCCCAGAAUAUGNN sense 868 1126 CAUAUUCUGGGAAGUAGUCNN antis 8681127 UCAACGACGAGAUCGAGCGNN sense 62 1128 CGCUCGAUCUCGUCGUUGANN antis 62

TABLE 4c GNAQ (rat and mouse): sense and antisensesequences with dTdT overhangs Numbering for target sequences is Rat GNAQNM_031036. SEQ Start of ID target NO SEQUENCE (5′-3′) Strand sequence1129 UAUUCCCACCUAGUCGACUdTdT sense 853 1130 AGUCGACUAGGUGGGAAUAdTdTantis 853 1131 UUCCCACCUAGUCGACUACdTdT sense 855 1132GUAGUCGACUAGGUGGGAAdTdT antis 855 1133 GCUUUUGAGAAUCCAUAUGdTdT sense 3671134 CAUAUGGAUUCUCAAAAGCdTdT antis 367 1135 CGGAGGAUCAACGACGAGAdTdTsense 55 1136 UCUCGUCGUUGAUCCUCCGdTdT antis 55 1137AUCUGACUCUACCAAAUACdTdT sense 459 1138 GUAUUUGGUAGAGUCAGAUdTdT antis 4591139 ACACAAUAAGGCUCAUGCAdTdT sense 312 1140 UGCAUGAGCCUUAUUGUGUdTdTantis 312 1141 AGGAUCAUCCACGGGUCGGdTdT sense 178 1142CCGACCCGUGGAUGAUCCUdTdT antis 178 1143 CCCAUACAAGUAUGAACACdTdT sense 2971144 GUGUUCAUACUUGUAUGGGdTdT antis 297 1145 CAAUAAGGCUCAUGCACAAdTdTsense 315 1146 UUGUGCAUGAGCCUUAUUGdTdT antis 315 1147AGGAUCAACGACGAGAUCGdTdT sense 58 1148 CGAUCUCGUCGUUGAUCCUdTdT antis 581149 UCAUGCACAAUUGGUUCGAdTdT sense 324 1150 UCGAACCAAUUGUGCAUGAdTdTantis 324 1151 GGAUCAACGACGAGAUCGAdTdT sense 59 1152UCGAUCUCGUCGUUGAUCCdTdT antis 59 1153 AGAGCUUGUGGAAUGAUCCdTdT sense 3981154 GGAUCAUUCCACAAGCUCUdTdT antis 398 1155 GAGGAUCAACGACGAGAUCdTdTsense 57 1156 GAUCUCGUCGUUGAUCCUCdTdT antis 57 1157GGAGGAUCAACGACGAGAUdTdT sense 56 1158 AUCUCGUCGUUGAUCCUCCdTdT antis 561159 UUUUGAGAAUCCAUAUGUAdTdT sense 369 1160 UACAUAUGGAUUCUCAAAAdTdTantis 369 1161 CAAGGAAGCCCGGAGGAUCdTdT sense 45 1162GAUCCUCCGGGCUUCCUUGdTdT antis 45 1163 UCUGACUCUACCAAAUACUdTdT sense 4601164 AGUAUUUGGUAGAGUCAGAdTdT antis 460 1165 AAGCGCGACGCCCGCCGGGdTdTsense 97 1166 CCCGGCGGGCGUCGCGCUUdTdT antis 97 1167ACAAUAAGGCUCAUGCACAdTdT sense 314 1168 UGUGCAUGAGCCUUAUUGUdTdT antis 3141169 UAAGGCUCAUGCACAAUUGdTdT sense 318 1170 CAAUUGUGCAUGAGCCUUAdTdTantis 318 1171 AAGCCCGGAGGAUCAACGAdTdT sense 50 1172UCGUUGAUCCUCCGGGCUUdTdT antis 50 1173 CUCAUGCACAAUUGGUUCGdTdT sense 3231174 CGAACCAAUUGUGCAUGAGdTdT antis 323 1175 UGCACAAUUGGUUCGAGAGdTdTsense 327 1176 CUCUCGAACCAAUUGUGCAdTdT antis 327 1177CACAAUUGGUUCGAGAGGUdTdT sense 329 1178 ACCUCUCGAACCAAUUGUGdTdT antis 3291179 CUAGUCGACUACUUCCCAGdTdT sense 862 1180 CUGGGAAGUAGUCGACUAGdTdTantis 862 1181 GCAGGGACAAGCGCGACGCdTdT sense 89 1182GCGUCGCGCUUGUCCCUGCdTdT antis 89 1183 UUGAGAAUCCAUAUGUAGAdTdT sense 3711184 UCUACAUAUGGAUUCUCAAdTdT antis 371 1185 GACUACUUCCCAGAAUAUGdTdTsense 868 1186 CAUAUUCUGGGAAGUAGUCdTdT antis 868 1187UCAACGACGAGAUCGAGCGdTdT sense 62 1188 CGCUCGAUCUCGUCGUUGAdTdT antis 62

TABLE 4d GNAQ dsRNA (rat and mouse): modified senseand antisense strands Numbering for target sequences is Rat GNAQNM_031036. Start of target SEQ SEQUENCE (5′-3′) Strand sequence ID NO:Modifications: Sense strand - all pyrimidines(U, C) are 2′OMe; antisense strand - pyrimidinesadjacent to A (UA, CA) are 2′Ome; 3′ end is dTdT uAuucccAccuAGucGAcudTdTsense 853 1189 AGUCGACuAGGUGGGAAuAdTdT antis 853 1190uucccAccuAGucGAcuAcdTdT sense 855 1191 GuAGUCGACuAGGUGGGAAdTdT antis 8551192 GcuuuuGAGAAuccAuAuGdTdT sense 367 1193 cAuAUGGAUUCUcAAAAGCdTdTantis 367 1194 cGGAGGAucAAcGAcGAGAdTdT sense 55 1195UCUCGUCGUUGAUCCUCCGdTdT antis 55 1196 AucuGAcucuAccAAAuAcdTdT sense 4591197 GuAUUUGGuAGAGUcAGAUdTdT antis 459 1198 AcAcAAuAAGGcucAuGcAdTdTsense 312 1199 UGcAUGAGCCUuAUUGUGUdTdT antis 312 1200AGGAucAuccAcGGGucGGdTdT sense 178 1201 CCGACCCGUGGAUGAUCCUdTdT antis 1781202 cccAuAcAAGuAuGAAcAcdTdT sense 297 1203 GUGUUcAuACUUGuAUGGGdTdTantis 297 1204 cAAuAAGGcucAuGcAcAAdTdT sense 315 1205UUGUGcAUGAGCCUuAUUGdTdT antis 315 1206 AGGAucAAcGAcGAGAucGdTdT sense 581207 CGAUCUCGUCGUUGAUCCUdTdT antis 58 1208 ucAuGcAcAAuuGGuucGAdTdT sense324 1209 UCGAACcAAUUGUGcAUGAdTdT antis 324 1210 GGAucAAcGAcGAGAucGAdTdTsense 59 1211 UCGAUCUCGUCGUUGAUCCdTdT antis 59 1212AGAGcuuGuGGAAuGAuccdTdT sense 398 1213 GGAUcAUUCcAcAAGCUCUdTdT antis 3981214 GAGGAucAAcGAcGAGAucdTdT sense 57 1215 GAUCUCGUCGUUGAUCCUCdTdT antis57 1216 GGAGGAucAAcGAcGAGAudTdT sense 56 1217 AUCUCGUCGUUGAUCCUCCdTdTantis 56 1218 uuuuGAGAAuccAuAuGuAdTdT sense 369 1219uAcAuAUGGAUUCUcAAAAdTdT antis 369 1220 cAAGGAAGcccGGAGGAucdTdT sense 451221 GAUCCUCCGGGCUUCCUUGdTdT antis 45 1222 ucuGAcucuAccAAAuAcudTdT sense460 1223 AGuAUUUGGuAGAGUcAGAdTdT antis 460 1224 AAGcGcGAcGcccGccGGGdTdTsense 97 1225 CCCGGCGGGCGUCGCGCUUdTdT antis 97 1226AcAAuAAGGcucAuGcAcAdTdT sense 314 1227 UGUGcAUGAGCCUuAUUGUdTdT antis 3141228 uAAGGcucAuGcAcAAuuGdTdT sense 318 1229 cAAUUGUGcAUGAGCCUuAdTdTantis 318 1230 AAGcccGGAGGAucAAcGAdTdT sense 50 1231UCGUUGAUCCUCCGGGCUUdTdT antis 50 1232 cucAuGcAcAAuuGGuucGdTdT sense 3231233 CGAACcAAUUGUGcAUGAGdTdT antis 323 1234 uGcAcAAuuGGuucGAGAGdTdTsense 327 1235 CUCUCGAACcAAUUGUGcAdTdT antis 327 1236cAcAAuuGGuucGAGAGGudTdT sense 329 1237 ACCUCUCGAACcAAUUGUGdTdT antis 3291238 cuAGucGAcuAcuucccAGdTdT sense 862 1239 CUGGGAAGuAGUCGACuAGdTdTantis 862 1240 GcAGGGAcAAGcGcGAcGcdTdT sense 89 1241GCGUCGCGCUUGUCCCUGCdTdT antis 89 1242 uuGAGAAuccAuAuGuAGAdTdT sense 3711243 UCuAcAuAUGGAUUCUcAAdTdT antis 371 1244 GAcuAcuucccAGAAuAuGdTdTsense 868 1245 cAuAUUCUGGGAAGuAGUCdTdT antis 868 1246ucAAcGAcGAGAucGAGcGdTdT sense 62 1247 CGCUCGAUCUCGUCGUUGAdTdT antis 621248 Modifications: Sense strand - all pyrimidines(U, C) are 2′OMe; antisense strand - pyrimidinesadjacent to A (UA, CA) are 2′Ome; 3′ end is thio (dTsdT)uAuucccAccuAGucGAcudTsdT sense 853 1249 AGUCGACuAGGUGGGAAuAdTsdT antis853 1250 uucccAccuAGucGAcuAcdTsdT sense 855 1251GuAGUCGACuAGGUGGGAAdTsdT antis 855 1252 GcuuuuGAGAAuccAuAuGdTsdT sense367 1253 cAuAUGGAUUCUcAAAAGCdTsdT antis 367 1254cGGAGGAucAAcGAcGAGAdTsdT sense 55 1255 UCUCGUCGUUGAUCCUCCGdTsdT antis 551256 AucuGAcucuAccAAAuAcdTsdT sense 459 1257 GuAUUUGGuAGAGUcAGAUdTsdTantis 459 1258 AcAcAAuAAGGcucAuGcAdTsdT sense 312 1259UGcAUGAGCCUuAUUGUGUdTsdT antis 312 1260 AGGAucAuccAcGGGucGGdTsdT sense178 1261 CCGACCCGUGGAUGAUCCUdTsdT antis 178 1262cccAuAcAAGuAuGAAcAcdTsdT sense 297 1263 GUGUUcAuACUUGuAUGGGdTsdT antis297 1264 cAAuAAGGcucAuGcAcAAdTsdT sense 315 1265UUGUGcAUGAGCCUuAUUGdTsdT antis 315 1266 AGGAucAAcGAcGAGAucGdTsdT sense58 1267 CGAUCUCGUCGUUGAUCCUdTsdT antis 58 1268 ucAuGcAcAAuuGGuucGAdTsdTsense 324 1269 UCGAACcAAUUGUGcAUGAdTsdT antis 324 1270GGAucAAcGAcGAGAucGAdTsdT sense 59 1271 UCGAUCUCGUCGUUGAUCCdTsdT antis 591272 AGAGcuuGuGGAAuGAuccdTsdT sense 398 1273 GGAUcAUUCcAcAAGCUCUdTsdTantis 398 1274 GAGGAucAAcGAcGAGAucdTsdT sense 57 1275GAUCUCGUCGUUGAUCCUCdTsdT antis 57 1276 GGAGGAucAAcGAcGAGAudTsdT sense 561277 AUCUCGUCGUUGAUCCUCCdTsdT antis 56 1278 uuuuGAGAAuccAuAuGuAdTsdTsense 369 1279 uAcAuAUGGAUUCUcAAAAdTsdT antis 369 1280cAAGGAAGcccGGAGGAucdTsdT sense 45 1281 GAUCCUCCGGGCUUCCUUGdTsdT antis 451282 ucuGAcucuAccAAAuAcudTsdT sense 460 1283 AGuAUUUGGuAGAGUcAGAdTsdTantis 460 1284 AAGcGcGAcGcccGccGGGdTsdT sense 97 1285CCCGGCGGGCGUCGCGCUUdTsdT antis 97 1286 AcAAuAAGGcucAuGcAcAdTsdT sense314 1287 UGUGcAUGAGCCUuAUUGUdTsdT antis 314 1288uAAGGcucAuGcAcAAuuGdTsdT sense 318 1289 cAAUUGUGcAUGAGCCUuAdTsdT antis318 1290 AAGcccGGAGGAucAAcGAdTsdT sense 50 1291 UCGUUGAUCCUCCGGGCUUdTsdTantis 50 1292 cucAuGcAcAAuuGGuucGdTsdT sense 323 1293CGAACcAAUUGUGcAUGAGdTsdT antis 323 1294 uGcAcAAuuGGuucGAGAGdTsdT sense327 1295 CUCUCGAACcAAUUGUGcAdTsdT antis 327 1296cAcAAuuGGuucGAGAGGudTsdT sense 329 1297 ACCUCUCGAACcAAUUGUGdTsdT antis329 1298 cuAGucGAcuAcuucccAGdTsdT sense 862 1299CUGGGAAGuAGUCGACuAGdTsdT antis 862 1300 GcAGGGAcAAGcGcGAcGcdTsdT sense89 1301 GCGUCGCGCUUGUCCCUGCdTsdT antis 89 1302 uuGAGAAuccAuAuGuAGAdTsdTsense 371 1303 UCuAcAuAUGGAUUCUcAAdTsdT antis 371 1304GAcuAcuucccAGAAuAuGdTsdT sense 868 1305 cAuAUUCUGGGAAGuAGUCdTsdT antis868 1306 ucAAcGAcGAGAucGAGcGdTsdT sense 62 1307 CGCUCGAUCUCGUCGUUGAdTsdTantis 62 1308 Modifications: Sense strand - all pyrimidines are2′OMe; antisense strand - pyrimidines adjacent to A (UA, CA) +U adjacent to another U (UU) or G (UG) are 2′Ome; 3′end is thio (dTsdT). uAuucccAccuAGucGAcudTsdT sense 853 1309AGUCGACuAGGuGGGAAuAdTsdT antis 853 1310 uucccAccuAGucGAcuAcdTsdT sense855 1311 GuAGUCGACuAGGuGGGAAdTsdT antis 855 1312GcuuuuGAGAAuccAuAuGdTsdT sense 367 1313 cAuAuGGAuUCUcAAAAGCdTsdT antis367 1314 cGGAGGAucAAcGAcGAGAdTsdT sense 55 1315 UCUCGUCGuuGAUCCUCCGdTsdTantis 55 1316 AucuGAcucuAccAAAuAcdTsdT sense 459 1317GuAuUuGGuAGAGUcAGAUdTsdT antis 459 1318 AcAcAAuAAGGcucAuGcAdTsdT sense312 1319 uGcAuGAGCCUuAuuGuGUdTsdT antis 312 1320AGGAucAuccAcGGGucGGdTsdT sense 178 1321 CCGACCCGuGGAuGAUCCUdTsdT antis178 1322 cccAuAcAAGuAuGAAcAcdTsdT sense 297 1323GuGuUcAuACuuGuAuGGGdTsdT antis 297 1324 cAAuAAGGcucAuGcAcAAdTsdT sense315 1325 uuGuGcAuGAGCCUuAuuGdTsdT antis 315 1326AGGAucAAcGAcGAGAucGdTsdT sense 58 1327 CGAUCUCGUCGuuGAUCCUdTsdT antis 581328 ucAuGcAcAAuuGGuucGAdTsdT sense 324 1329 UCGAACcAAuuGuGcAuGAdTsdTantis 324 1330 GGAucAAcGAcGAGAucGAdTsdT sense 59 1331UCGAUCUCGUCGuuGAUCCdTsdT antis 59 1332 AGAGcuuGuGGAAuGAuccdTsdT sense398 1333 GGAUcAuUCcAcAAGCUCUdTsdT antis 398 1334GAGGAucAAcGAcGAGAucdTsdT sense 57 1335 GAUCUCGUCGuuGAUCCUCdTsdT antis 571336 GGAGGAucAAcGAcGAGAudTsdT sense 56 1337 AUCUCGUCGuuGAUCCUCCdTsdTantis 56 1338 uuuuGAGAAuccAuAuGuAdTsdT sense 369 1339uAcAuAuGGAuUCUcAAAAdTsdT antis 369 1340 cAAGGAAGcccGGAGGAucdTsdT sense45 1341 GAUCCUCCGGGCuUCCuuGdTsdT antis 45 1342 ucuGAcucuAccAAAuAcudTsdTsense 460 1343 AGuAuUuGGuAGAGUcAGAdTsdT antis 460 1344AAGcGcGAcGcccGccGGGdTsdT sense 97 1345 CCCGGCGGGCGUCGCGCuUdTsdT antis 971346 AcAAuAAGGcucAuGcAcAdTsdT sense 314 1347 uGuGcAuGAGCCUuAuuGUdTsdTantis 314 1348 uAAGGcucAuGcAcAAuuGdTsdT sense 318 1349cAAuuGuGcAuGAGCCUuAdTsdT antis 318 1350 AAGcccGGAGGAucAAcGAdTsdT sense50 1351 UCGuuGAUCCUCCGGGCuUdTsdT antis 50 1352 cucAuGcAcAAuuGGuucGdTsdTsense 323 1353 CGAACcAAuuGuGcAuGAGdTsdT antis 323 1354uGcAcAAuuGGuucGAGAGdTsdT sense 327 1355 CUCUCGAACcAAuuGuGcAdTsdT antis327 1356 cAcAAuuGGuucGAGAGGudTsdT sense 329 1357ACCUCUCGAACcAAuuGuGdTsdT antis 329 1358 cuAGucGAcuAcuucccAGdTsdT sense862 1359 CuGGGAAGuAGUCGACuAGdTsdT antis 862 1360GcAGGGAcAAGcGcGAcGcdTsdT sense 89 1361 GCGUCGCGCuuGUCCCuGCdTsdT antis 891362 uuGAGAAuccAuAuGuAGAdTsdT sense 371 1363 UCuAcAuAuGGAuUCUcAAdTsdTantis 371 1364 GAcuAcuucccAGAAuAuGdTsdT sense 868 1365cAuAuUCuGGGAAGuAGUCdTsdT antis 868 1366 ucAAcGAcGAGAucGAGcGdTsdT sense62 1367 CGCUCGAUCUCGUCGuuGAdTsdT antis 62 1368

Example 3 In Vitro Screening

For in vitro screening, cells expressing GNAQ were utilized. Someexemplary cell lines expressing GNAQ include, but are not limited to,human melanoma cell lines OMM1.3 and MEL 285, and Mel 202. OMM1.3 areliver metastisis cells that include a mutant GNAQ gene. MEL285 areprimary uveal melanoma cells that include a WT GNAQ gene. MEL202 arealso primary uveal melanoma but include a mutant GNAQ gene. A549 (lungcarcinoma) and A375 (malignant melanoma) are cancer cell linesexpressing WT GNAQ.

Cells expressing human GNAQ with the activating GNAQ mutation wereobtained following the method outlined in PCT publication numberWO2008/098208, which is incorporated herein in its entirety for allpurposes.

The dsRNAs were screened for in vitro inhibition of the target gene.Tissue culture cells were transfected with the dsRNA. Target gene mRNAlevels were assayed using qPCR (real time PCR).

Cell Culture and Transfections:

A549, A375, OMM1.3 and UMEL202 cells were grown to near confluence at37° C. in an atmosphere of 5% CO₂ in specific medium (ATCC) supplementedwith 10% FBS, streptomycin, and glutamine (ATCC) before being releasedfrom the plate by trypsinization. Reverse transfection was carried outby adding 5 μl of Opti-MEM to 5 μl of siRNA duplexes (Tables 5-7) perwell into a 96-well plate along with 10 μl of Opti-MEM plus 0.2 μl ofLipofectamine RNAiMax per well (Invitrogen, Carlsbad Calif. cat#13778-150) and incubated at room temperature for 15 minutes. 80 μl ofcomplete growth media without antibiotics containing 2×10⁴ cells werethen added. Cells were incubated for 24 hours prior to RNA purification.Single dose experiments were performed at either 0.1 nM, 1.0 nM, or and10.0 nM final duplex concentration and dose response experiments weredone with 10, 1.66, 0.27, 0.046, 0.0077, 0.0012, 0.00021, 0.000035 nM ofselected duplexes.

TABLE 5 Duplex (dsRNA) names and corresponding sample names Sample nameDuplex Name ssRNA name 1 AD-20032 36864 36865 2 AD-20033 36866 36867 3AD-20034 36868 36869 4 AD-20035 36870 36871 5 AD-20036 36872 36873 6AD-20037 36874 36875 7 AD-20038 36876 36877 8 AD-20039 36878 36879 9AD-20040 36880 36881 10 AD-20041 36882 36883 11 AD-20042 36884 36885 12AD-20043 36886 36887 13 AD-20044 36888 36889 14 AD-20045 36890 36891 15AD-20046 36892 36893 16 AD-20047 36894 36895 17 AD-20048 36896 36897 18AD-20049 36898 36899 19 AD-20050 36900 36901 20 AD-20051 36902 36903 21AD-20052 36904 36905 22 AD-20053 36906 36907 23 AD-20054 36910 36911 24AD-20055 36912 36913 25 AD-20056 36914 36915 26 AD-20057 36916 36917 27AD-20058 36918 36919 28 AD-20059 36920 36921 29 AD-20060 36922 36923 30AD-20061 36924 36925 31 AD-20062 36926 36927 32 AD-20063 36928 36929 33AD-20064 36930 36931 34 AD-20065 36932 36933 35 AD-20066 36934 36935 36AD-20067 36936 36937 37 AD-20068 36938 36939 38 AD-20069 36940 36941 39AD-20070 36942 36943 40 AD-20071 36946 36947 41 AD-20072 36948 36949 42AD-20073 36950 36951 43 AD-20074 36954 36955 87 AD-20075 36956 36957 44AD-20076 36958 36959 45 AD-20077 36960 36961 46 AD-20078 36962 36963 47AD-20079 36964 36965 48 AD-20080 36966 36967 49 AD-20081 36968 36969 50AD-20082 36970 36971 51 AD-20083 36972 36973 52 AD-20084 36974 36975 53AD-20085 36976 36977 54 AD-20086 36978 36979 55 AD-20087 36980 36981 56AD-20088 36982 36983 57 AD-20089 36984 36985 58 AD-20090 36986 36987 59AD-20091 36988 36989 60 AD-20092 36990 36991 61 AD-20093 36992 36993 62AD-20094 36994 36995 63 AD-20095 36996 36997 64 AD-20096 36998 36999 65AD-20097 37000 37001 88 AD-20098 37002 37003 66 AD-20099 37004 37005 67AD-20100 37006 37007 68 AD-20101 37008 37009 69 AD-20102 37010 37011 89AD-20103 37012 37013 70 AD-20104 37014 37015 95 AD-20105 37016 37017 71AD-20106 37018 37019 72 AD-20107 37022 37023 73 AD-20108 37024 37025 74AD-20109 37026 37027 75 AD-20110 37032 37033 76 AD-20111 37034 37035 77AD-20112 37036 37037 78 AD-20113 37038 37039 79 AD-20114 37040 37041 80AD-20115 37042 37043 81 AD-20116 37044 37045 82 AD-20117 37046 37047 83AD-20118 37048 37049 84 AD-20119 37050 37051 85 AD-20120 37052 37053 86AD-20121 37054 37055 91 AD-20193 36908 36909 92 AD-20194 36945 36944 93AD-20195 37020 37021 94 AD-20196 37028 37029 95 AD-20197 37030 37031

TABLE 6 Sequences of dsRNA targeting Human GNAQ (NM_002072.2)(target is position of 5′ base on transcript of NM_002072.2 SEQ SEQDuplex ID Unmodified sequence ID Modified sequence name Strand TargetNO: 5′ to 3′ NO: 5′ to 3′ AD-20032 S 1215 1369 UACUAAUUUAUUGCCGUCC 1527uAcuAAuuuAuuGccGuccdTdT A 1215 1370 GGACGGCAAUAAAUUAGUA 1528GGACGGcAAuAAAUuAGuAdTdT AD-20033 S 1217 1371 CUAAUUUAUUGCCGUCCUG 1529cuAAuuuAuuGccGuccuGdTdT A 1217 1372 CAGGACGGCAAUAAAUUAG 1530cAGGACGGcAAuAAAUuAGdTdT AD-20034 S 1216 1373 ACUAAUUUAUUGCCGUCCU 1531AcuAAuuuAuuGccGuccudTdT A 1216 1374 AGGACGGCAAUAAAUUAGU 1532AGGACGGcAAuAAAUuAGUdTdT AD-20035 S 1322 1375 GUACAGUCCCAGCACAUUU 1533GuAcAGucccAGcAcAuuudTdT A 1322 1376 AAAUGUGCUGGGACUGUAC 1534AAAUGUGCUGGGACUGuACdTdT AD-20036 S 1220 1377 AUUUAUUGCCGUCCUGGAC 1535AuuuAuuGccGuccuGGAcdTdT A 1220 1378 GUCCAGGACGGCAAUAAAU 1536GUCcAGGACGGcAAuAAAUdTdT AD-20037 S 1265 1379 GUAGUAAAUAUUAUGAUUU 1537GuAGuAAAuAuuAuGAuuudTdT A 1265 1380 AAAUCAUAAUAUUUACUAC 1538AAAUcAuAAuAUUuACuACdTdT AD-20038 S 1218 1381 UAAUUUAUUGCCGUCCUGG 1539uAAuuuAuuGccGuccuGGdTdT A 1218 1382 CCAGGACGGCAAUAAAUUA 1540CcAGGACGGcAAuAAAUuAdTdT AD-20039 S 1175 1383 ACAAGAGGGACUGUAUUUC 1541AcAAGAGGGAcuGuAuuucdTdT A 1175 1384 GAAAUACAGUCCCUCUUGU 1542GAAAuAcAGUCCCUCUUGUdTdT AD-20040 S 1223 1385 UAUUGCCGUCCUGGACUCU 1543uAuuGccGuccuGGAcucudTdT A 1223 1386 AGAGUCCAGGACGGCAAUA 1544AGAGUCcAGGACGGcAAuAdTdT AD-20041 S 1319 1387 GAAGUACAGUCCCAGCACA 1545GAAGuAcAGucccAGcAcAdTdT A 1319 1388 UGUGCUGGGACUGUACUUC 1546UGUGCUGGGACUGuACUUCdTdT AD-20042 S 1285 1389 AUUUAAACUAUUCAGAGGA 1547AuuuAAAcuAuucAGAGGAdTdT A 1285 1390 UCCUCUGAAUAGUUUAAAU 1548UCCUCUGAAuAGUUuAAAUdTdT AD-20043 S 1213 1391 AAUACUAAUUUAUUGCCGU 1549AAuAcuAAuuuAuuGccGudTdT A 1213 1392 ACGGCAAUAAAUUAGUAUU 1550ACGGcAAuAAAUuAGuAUUdTdT AD-20044 S 1810 1393 CAGCCAUAGCUUGAUUGCU 1551cAGccAuAGcuuGAuuGcudTdT A 1810 1394 AGCAAUCAAGCUAUGGCUG 1552AGcAAUcAAGCuAUGGCUGdTdT AD-20045 S 1590 1395 GUCAGGACACAUCGUUCGA 1553GucAGGAcAcAucGuucGAdTdT A 1590 1396 UCGAACGAUGUGUCCUGAC 1554UCGAACGAUGUGUCCUGACdTdT AD-20046 S 1149 1397 CUUCCCUGGUGGGCUAUUG 1555cuucccuGGuGGGcuAuuGdTdT A 1149 1398 CAAUAGCCCACCAGGGAAG 1556cAAuAGCCcACcAGGGAAGdTdT AD-20047 S 1971 1399 GACACUACAUUACCCUAAU 1557GAcAcuAcAuuAcccuAAudTdT A 1971 1400 AUUAGGGUAAUGUAGUGUC 1558AUuAGGGuAAUGuAGUGUCdTdT AD-20048 S 1237 1401 ACUCUGUGUGAGCGUGUCC 1559AcucuGuGuGAGcGuGuccdTdT A 1237 1402 GGACACGCUCACACAGAGU 1560GGAcACGCUcAcAcAGAGUdTdT AD-20049 S 1152 1403 CCCUGGUGGGCUAUUGAAG 1561cccuGGuGGGcuAuuGAAGdTdT A 1152 1404 CUUCAAUAGCCCACCAGGG 1562CUUcAAuAGCCcACcAGGGdTdT AD-20050 S 1575 1405 CUCUCAAAUGAUACAGUCA 1563cucucAAAuGAuAcAGucAdTdT A 1575 1406 UGACUGUAUCAUUUGAGAG 1564UGACUGuAUcAUUUGAGAGdTdT AD-20051 S 1105 1407 AGUACAAUCUGGUCUAAUU 1565AGuAcAAucuGGucuAAuudTdT A 1105 1408 AAUUAGACCAGAUUGUACU 1566AAUuAGACcAGAUUGuACUdTdT AD-20052 S 1407 1409 CACAAAGAUAAGACUUGUU 1567cAcAAAGAuAAGAcuuGuudTdT A 1407 1410 AACAAGUCUUAUCUUUGUG 1568AAcAAGUCUuAUCUUUGUGdTdT AD-20053 S 1108 1411 ACAAUCUGGUCUAAUUGUG 1569AcAAucuGGucuAAuuGuGdTdT A 1108 1412 CACAAUUAGACCAGAUUGU 1570cAcAAUuAGACcAGAUUGUdTdT AD-20193 S 1395 1413 CAGUCAUGCACUCACAAAG 1571cAGucAuGcAcucAcAAAGdTdT A 1395 1414 CUUUGUGAGUGCAUGACUG 1572CUUUGUGAGUGcAUGACUGdTdT AD-20054 S 1595 1415 GACACAUCGUUCGAUUUAA 1573GAcAcAucGuucGAuuuAAdTdT A 1595 1416 UUAAAUCGAACGAUGUGUC 1574UuAAAUCGAACGAUGUGUCdTdT AD-20055 S 1992 1417 CUGCUACCCAGAACCUUUU 1575cuGcuAcccAGAAccuuuudTdT A 1992 1418 AAAAGGUUCUGGGUAGCAG 1576AAAAGGUUCUGGGuAGcAGdTdT AD-20056 S 1809 1419 UCAGCCAUAGCUUGAUUGC 1577ucAGccAuAGcuuGAuuGcdTdT A 1809 1420 GCAAUCAAGCUAUGGCUGA 1578GcAAUcAAGCuAUGGCUGAdTdT AD-20057 S 1203 1421 CAAUUUGCAUAAUACUAAU 1579cAAuuuGcAuAAuAcuAAudTdT A 1203 1422 AUUAGUAUUAUGCAAAUUG 1580AUuAGuAUuAUGcAAAUUGdTdT AD-20058 S 1804 1423 UACCUUCAGCCAUAGCUUG 1581uAccuucAGccAuAGcuuGdTdT A 1804 1424 CAAGCUAUGGCUGAAGGUA 1582cAAGCuAUGGCUGAAGGuAdTdT AD-20059 S 1968 1425 ACAGACACUACAUUACCCU 1583AcAGAcAcuAcAuuAcccudTdT A 1968 1426 AGGGUAAUGUAGUGUCUGU 1584AGGGuAAUGuAGUGUCUGUdTdT AD-20060 S 1214 1427 AUACUAAUUUAUUGCCGUC 1585AuAcuAAuuuAuuGccGucdTdT A 1214 1428 GACGGCAAUAAAUUAGUAU 1586GACGGcAAuAAAUuAGuAUdTdT AD-20061 S 1159 1429 GGGCUAUUGAAGAUACACA 1587GGGcuAuuGAAGAuAcAcAdTdT A 1159 1430 UGUGUAUCUUCAAUAGCCC 1588UGUGuAUCUUcAAuAGCCCdTdT AD-20062 S 1603 1431 GUUCGAUUUAAGCCAUCAU 1589GuucGAuuuAAGccAucAudTdT A 1603 1432 AUGAUGGCUUAAAUCGAAC 1590AUGAUGGCUuAAAUCGAACdTdT AD-20063 S 1123 1433 UGUGCCUCCUAGACACCCG 1591uGuGccuccuAGAcAcccGdTdT A 1123 1434 CGGGUGUCUAGGAGGCACA 1592CGGGUGUCuAGGAGGcAcAdTdT AD-20064 S 1233 1435 CUGGACUCUGUGUGAGCGU 1593cuGGAcucuGuGuGAGcGudTdT A 1233 1436 ACGCUCACACAGAGUCCAG 1594ACGCUcAcAcAGAGUCcAGdTdT AD-20065 S 1930 1437 ACCCUCUCUUUCAAUUGCA 1595AcccucucuuucAAuuGcAdTdT A 1930 1438 UGCAAUUGAAAGAGAGGGU 1596UGcAAUUGAAAGAGAGGGUdTdT AD-20066 S 1969 1439 CAGACACUACAUUACCCUA 1597cAGAcAcuAcAuuAcccuAdTdT A 1969 1440 UAGGGUAAUGUAGUGUCUG 1598uAGGGuAAUGuAGUGUCUGdTdT AD-20067 S 1219 1441 AAUUUAUUGCCGUCCUGGA 1599AAuuuAuuGccGuccuGGAdTdT A 1219 1442 UCCAGGACGGCAAUAAAUU 1600UCcAGGACGGcAAuAAAUUdTdT AD-20068 S 1241 1443 UGUGUGAGCGUGUCCACAG 1601uGuGuGAGcGuGuccAcAGdTdT A 1241 1444 CUGUGGACACGCUCACACA 1602CUGUGGAcACGCUcAcAcAdTdT AD-20069 S 1153 1445 CCUGGUGGGCUAUUGAAGA 1603ccuGGuGGGcuAuuGAAGAdTdT A 1153 1446 UCUUCAAUAGCCCACCAGG 1604UCUUcAAuAGCCcACcAGGdTdT AD-20070 S 1805 1447 ACCUUCAGCCAUAGCUUGA 1605AccuucAGccAuAGcuuGAdTdT A 1805 1448 UCAAGCUAUGGCUGAAGGU 1606UcAAGCuAUGGCUGAAGGUdTdT AD-20194 S 1312 1449 GGAUGCUGAAGUACAGUCC 1607GGAuGcuGAAGuAcAGuccdTdT A 1312 1450 GGACUGUACUUCAGCAUCC 1608GGACUGuACUUcAGcAUCCdTdT AD-20071 S 1546 1451 AUCCUAGUUCCAUUCUUGG 1609AuccuAGuuccAuucuuGGdTdT A 1546 1452 CCAAGAAUGGAACUAGGAU 1610CcAAGAAUGGAACuAGGAUdTdT AD-20072 S 1547 1453 UCCUAGUUCCAUUCUUGGU 1611uccuAGuuccAuucuuGGudTdT A 1547 1454 ACCAAGAAUGGAACUAGGA 1612ACcAAGAAUGGAACuAGGAdTdT AD-20073 S 1103 1455 GGAGUACAAUCUGGUCUAA 1613GGAGuAcAAucuGGucuAAdTdT A 1103 1456 UUAGACCAGAUUGUACUCC 1614UuAGACcAGAUUGuACUCCdTdT A 1334 1457 CACAUUUCCUCUCUAUCUU 1615cAcAuuuccucucuAucuudTdT A 1334 1458 AAGAUAGAGAGGAAAUGUG 1616AAGAuAGAGAGGAAAUGUGdTdT AD-20074 S 1255 1459 CACAGAGUUUGUAGUAAAU 1617cAcAGAGuuuGuAGuAAAudTdT A 1255 1460 AUUUACUACAAACUCUGUG 1618AUUuACuAcAAACUCUGUGdTdT AD-20075 S 1967 1461 AACAGACACUACAUUACCC 1619AAcAGAcAcuAcAuuAcccdTdT A 1967 1462 GGGUAAUGUAGUGUCUGUU 1620GGGuAAUGuAGUGUCUGUUdTdT AD-20076 S 1391 1463 UUCUCAGUCAUGCACUCAC 1621uucucAGucAuGcAcucAcdTdT A 1391 1464 GUGAGUGCAUGACUGAGAA 1622GUGAGUGcAUGACUGAGAAdTdT AD-20077 S 1124 1465 GUGCCUCCUAGACACCCGC 1623GuGccuccuAGAcAcccGcdTdT A 1124 1466 GCGGGUGUCUAGGAGGCAC 1624GCGGGUGUCuAGGAGGcACdTdT AD-20078 S 1612 1467 AAGCCAUCAUCAGCUUAAU 1625AAGccAucAucAGcuuAAudTdT A 1612 1468 AUUAAGCUGAUGAUGGCUU 1626AUuAAGCUGAUGAUGGCUUdTdT AD-20079 S 1933 1469 CUCUCUUUCAAUUGCAGAU 1627cucucuuucAAuuGcAGAudTdT A 1933 1470 AUCUGCAAUUGAAAGAGAG 1628AUCUGcAAUUGAAAGAGAGdTdT AD-20080 S 1078 1471 ACACCAUCCUCCAGUUGAA 1629AcAccAuccuccAGuuGAAdTdT A 1078 1472 UUCAACUGGAGGAUGGUGU 1630UUcAACUGGAGGAUGGUGUdTdT AD-20081 S 1545 1473 UAUCCUAGUUCCAUUCUUG 1631uAuccuAGuuccAuucuuGdTdT A 1545 1474 CAAGAAUGGAACUAGGAUA 1632cAAGAAUGGAACuAGGAuAdTdT AD-20082 S 1109 1475 CAAUCUGGUCUAAUUGUGC 1633cAAucuGGucuAAuuGuGcdTdT A 1109 1476 GCACAAUUAGACCAGAUUG 1634GcAcAAUuAGACcAGAUUGdTdT AD-20083 S 1398 1477 UCAUGCACUCACAAAGAUA 1635ucAuGcAcucAcAAAGAuAdTdT A 1398 1478 UAUCUUUGUGAGUGCAUGA 1636uAUCUUUGUGAGUGcAUGAdTdT AD-20084 S 1970 1479 AGACACUACAUUACCCUAA 1637AGAcAcuAcAuuAcccuAAdTdT A 1970 1480 UUAGGGUAAUGUAGUGUCU 1638UuAGGGuAAUGuAGUGUCUdTdT AD-20085 S 1173 1481 ACACAAGAGGGACUGUAUU 1639AcAcAAGAGGGAcuGuAuudTdT A 1173 1482 AAUACAGUCCCUCUUGUGU 1640AAuAcAGUCCCUCUUGUGUdTdT AD-20086 S 1313 1483 GAUGCUGAAGUACAGUCCC 1641GAuGcuGAAGuAcAGucccdTdT A 1313 1484 GGGACUGUACUUCAGCAUC 1642GGGACUGuACUUcAGcAUCdTdT AD-20087 S 1811 1485 AGCCAUAGCUUGAUUGCUC 1643AGccAuAGcuuGAuuGcucdTdT A 1811 1486 GAGCAAUCAAGCUAUGGCU 1644GAGcAAUcAAGCuAUGGCUdTdT AD-20088 S 1862 1487 CACAGGAGUCCUUUCUUUU 1645cAcAGGAGuccuuucuuuudTdT A 1862 1488 AAAAGAAAGGACUCCUGUG 1646AAAAGAAAGGACUCCUGUGdTdT AD-20089 S 1600 1489 AUCGUUCGAUUUAAGCCAU 1647AucGuucGAuuuAAGccAudTdT A 1600 1490 AUGGCUUAAAUCGAACGAU 1648AUGGCUuAAAUCGAACGAUdTdT AD-20090 S 1618 1491 UCAUCAGCUUAAUUUAAGU 1649ucAucAGcuuAAuuuAAGudTdT A 1618 1492 ACUUAAAUUAAGCUGAUGA 1650ACUuAAAUuAAGCUGAUGAdTdT AD-20091 S 1332 1493 AGCACAUUUCCUCUCUAUC 1651AGcAcAuuuccucucuAucdTdT A 1332 1494 GAUAGAGAGGAAAUGUGCU 1652GAuAGAGAGGAAAUGUGCUdTdT AD-20092 S 1157 1495 GUGGGCUAUUGAAGAUACA 1653GuGGGcuAuuGAAGAuAcAdTdT A 1157 1496 UGUAUCUUCAAUAGCCCAC 1654UGuAUCUUcAAuAGCCcACdTdT AD-20093 S 888 1497 AUCAUGUAUUCCCAUCUAG 1655AucAuGuAuucccAucuAGdTdT A 888 1498 CUAGAUGGGAAUACAUGAU 1656CuAGAUGGGAAuAcAUGAUdTdT AD-20094 S 1855 1499 AAAGACACACAGGAGUCCU 1657AAAGAcAcAcAGGAGuccudTdT A 1855 1500 AGGACUCCUGUGUGUCUUU 1658AGGACUCCUGUGUGUCUUUdTdT AD-20095 S 1579 1501 CAAAUGAUACAGUCAGGAC 1659cAAAuGAuAcAGucAGGAcdTdT A 1579 1502 GUCCUGACUGUAUCAUUUG 1660GUCCUGACUGuAUcAUUUGdTdT AD-20096 S 805 1503 UUAGAACAAUUAUCACAUA 1661uuAGAAcAAuuAucAcAuAdTdT A 805 1504 UAUGUGAUAAUUGUUCUAA 1662uAUGUGAuAAUUGUUCuAAdTdT AD-20097 S 1554 1505 UCCAUUCUUGGUCAAGUUU 1663uccAuucuuGGucAAGuuudTdT A 1554 1506 AAACUUGACCAAGAAUGGA 1664AAACUUGACcAAGAAUGGAdTdT AD-20098 S 1113 1507 CUGGUCUAAUUGUGCCUCC 1665cuGGucuAAuuGuGccuccdTdT A 1113 1508 GGAGGCACAAUUAGACCAG 1666GGAGGcAcAAUuAGACcAGdTdT AD-20099 S 1174 1509 CACAAGAGGGACUGUAUUU 1667cAcAAGAGGGAcuGuAuuudTdT A 1174 1510 AAAUACAGUCCCUCUUGUG 1668AAAuAcAGUCCCUCUUGUGdTdT AD-20100 S 1735 1511 UCUUGUCUCACUUUGGACU 1669ucuuGucucAcuuuGGAcudTdT A 1735 1512 AGUCCAAAGUGAGACAAGA 1670AGUCcAAAGUGAGAcAAGAdTdT AD-20101 S 1450 1513 UUUUCUAUGGAGCAAAACA 1671uuuucuAuGGAGcAAAAcAdTdT A 1450 1514 UGUUUUGCUCCAUAGAAAA 1672UGUUUUGCUCcAuAGAAAAdTdT AD-20102 S 804 1515 UUUAGAACAAUUAUCACAU 1673uuuAGAAcAAuuAucAcAudTdT A 804 1516 AUGUGAUAAUUGUUCUAAA 1674AUGUGAuAAUUGUUCuAAAdTdT AD-20103 S 1866 1517 GGAGUCCUUUCUUUUGAAA 1675GGAGuccuuucuuuuGAAAdTdT A 1866 1518 UUUCAAAAGAAAGGACUCC 1676UUUcAAAAGAAAGGACUCCdTdT AD-20104 S 1610 1519 UUAAGCCAUCAUCAGCUUA 1677uuAAGccAucAucAGcuuAdTdT A 1610 1520 UAAGCUGAUGAUGGCUUAA 1678uAAGCUGAUGAUGGCUuAAdTdT AD-20105 S 1117 1521 UCUAAUUGUGCCUCCUAGA 1679ucuAAuuGuGccuccuAGAdTdT A 1117 1522 UCUAGGAGGCACAAUUAGA 1680UCuAGGAGGcAcAAUuAGAdTdT AD-20106 S 1320 1523 AAGUACAGUCCCAGCACAU 1681AAGuAcAGucccAGcAcAudTdT A 1320 1524 AUGUGCUGGGACUGUACUU 1682AUGUGCUGGGACUGuACUUdTdT AD-20195 S 1317 1525 CUGAAGUACAGUCCCAGCA 1683cuGAAGuAcAGucccAGcAdTdT A 1317 1526 UGCUGGGACUGUACUUCAG 1684UGCUGGGACUGuACUUcAGdTdT

TABLE 7a Sequences of dsRNA targeting Mouse GNAQ (NM_031036)(target is position of 5′ base on transcript of NM_031036 SEQ SEQ DuplexID Unmodified sequence ID Modified sequence Name Strand Target NO: 5′to 3′ NO: 5′ to 3′ AD-20107 S 853 1685 UAUUCCCACCUAGUCGACU 1719uAuucccAccuAGucGAcudTdT A 853 1686 AGUCGACUAGGUGGGAAUA 1720AGUCGACuAGGUGGGAAuAdTdT AD-20108 S 855 1687 UUCCCACCUAGUCGACUAC 1721uucccAccuAGucGAcuAcdTdT A 855 1688 GUAGUCGACUAGGUGGGAA 1722GuAGUCGACuAGGUGGGAAdTdT AD-20109 S 367 1689 GCUUUUGAGAAUCCAUAUG 1723GcuuuuGAGAAuccAuAuGdTdT A 367 1690 CAUAUGGAUUCUCAAAAGC 1724cAuAUGGAUUCUcAAAAGCdTdT AD-20196 S 55 1691 CGGAGGAUCAACGACGAGA 1725cGGAGGAucAAcGAcGAGAdTdT A 55 1692 UCUCGUCGUUGAUCCUCCG 1726UCUCGUCGUUGAUCCUCCGdTdT AD-20197 S 459 1693 AUCUGACUCUACCAAAUAC 1727AucuGAcucuAccAAAuAcdTdT A 459 1694 GUAUUUGGUAGAGUCAGAU 1728GuAUUUGGuAGAGUcAGAUdTdT AD-20110 S 312 1695 ACACAAUAAGGCUCAUGCA 1729AcAcAAuAAGGcucAuGcAdTdT A 312 1696 UGCAUGAGCCUUAUUGUGU 1730UGcAUGAGCCUuAUUGUGUdTdT AD-20111 S 178 1697 AGGAUCAUCCACGGGUCGG 1731AGGAucAuccAcGGGucGGdTdT A 178 1698 CCGACCCGUGGAUGAUCCU 1732CCGACCCGUGGAUGAUCCUdTdT AD-20112 S 297 1699 CCCAUACAAGUAUGAACAC 1733cccAuAcAAGuAuGAAcAcdTdT A 297 1700 GUGUUCAUACUUGUAUGGG 1734GUGUUcAuACUUGuAUGGGdTdT AD-20113 S 315 1701 CAAUAAGGCUCAUGCACAA 1735cAAuAAGGcucAuGcAcAAdTdT A 315 1702 UUGUGCAUGAGCCUUAUUG 1736UUGUGcAUGAGCCUuAUUGdTdT AD-20114 S 58 1703 AGGAUCAACGACGAGAUCG 1737AGGAucAAcGAcGAGAucGdTdT A 58 1704 CGAUCUCGUCGUUGAUCCU 1738CGAUCUCGUCGUUGAUCCUdTdT AD-20115 S 324 1705 UCAUGCACAAUUGGUUCGA 1739ucAuGcAcAAuuGGuucGAdTdT A 324 1706 UCGAACCAAUUGUGCAUGA 1740UCGAACcAAUUGUGcAUGAdTdT AD-20116 S 59 1707 GGAUCAACGACGAGAUCGA 1741GGAucAAcGAcGAGAucGAdTdT A 59 1708 UCGAUCUCGUCGUUGAUCC 1742UCGAUCUCGUCGUUGAUCCdTdT AD-20117 S 398 1709 AGAGCUUGUGGAAUGAUCC 1743AGAGcuuGuGGAAuGAuccdTdT A 398 1710 GGAUCAUUCCACAAGCUCU 1744GGAUcAUUCcAcAAGCUCUdTdT AD-20118 S 57 1711 GAGGAUCAACGACGAGAUC 1745GAGGAucAAcGAcGAGAucdTdT A 57 1712 GAUCUCGUCGUUGAUCCUC 1746GAUCUCGUCGUUGAUCCUCdTdT AD-20119 S 56 1713 GGAGGAUCAACGACGAGAU 1747GGAGGAucAAcGAcGAGAudTdT A 56 1714 AUCUCGUCGUUGAUCCUCC 1748AUCUCGUCGUUGAUCCUCCdTdT AD-20120 S 369 1715 UUUUGAGAAUCCAUAUGUA 1749uuuuGAGAAuccAuAuGuAdTdT A 369 1716 UACAUAUGGAUUCUCAAAA 1750uAcAuAUGGAUUCUcAAAAdTdT AD-20121 S 45 1717 CAAGGAAGCCCGGAGGAUC 1751cAAGGAAGcccGGAGGAucdTdT A 45 1718 GAUCCUCCGGGCUUCCUUG 1752GAUCCUCCGGGCUUCCUUGdTdT

TABLE 7b Sequences of dsRNA targeting GNAQ (AD-20196 and AD-20197 only)SEQ SEQ Duplex ID Unmodified sequence ID Modified sequence Name StrandNO: 5′ to 3′ NO: 5′ to 3′ AD-20196 S 1753 CGGAGGAUCAACGACGAGA 1757cGGAGGAucAAcGAcGAGAdTdT A 1754 UCUCGUCGUUGAUCCUCCG 1758UCUCGUCGUUGAUCCUCCGdTdT AD-20197 S 1755 AUCUGACUCUACCAAAUAC 1759AucuGAcucuAccAAAuAcdTdT A 1756 GUAUUUGGUAGAGUCAGAU 1760GuAUUUGGuAGAGUcAGAUdTdT

Total RNA Isolation using MagMAX-96 Total RNA Isolation Kit (AppliedBiosystem, Foster City Calif., Part #: AM1830):

Cells were harvested and lysed in 140 μl of Lysis/Binding Solution thenmixed for 1 minute at 850 rpm using and Eppendorf Thermomixer (themixing speed was the same throughout the process). Twenty micro litersof magnetic beads were added into cell-lysate and mixed for 5 minutes.Magnetic beads were captured using magnetic stand and the supernatantwas removed without disturbing the beads. After removing supernatant,magnetic beads were washed with Wash Solution 1 (isopropanol added) andmixed for 1 minute. Beads were capture again and supernatant removed.Beads were then washed with 150 μl Wash Solution 2 (Ethanol added),captured and supernatant was removed. 50 ul of DNase mixture (MagMaxturbo DNase Buffer and Turbo DNase) was then added to the beads and theywere mixed for 10 to 15 minutes. After mixing, 100 μl of RNA RebindingSolution was added and mixed for 3 minutes. Supernatant was removed andmagnetic beads were washed again with 150 μl Wash Solution 2 and mixedfor 1 minute and supernatant was removed completely. The magnetic beadswere mixed for 2 minutes to dry before RNA it was eluted with 50 μl ofwater.

Total RNA Isolation Using RNAqueous®-96 Well Plate Procedure (AppliedBiosystem, Foster City Calif., Part #: 1812):

Cells were lysed for 5 minutes in 200 μl of Lysis/Binding Solution. 100μl of 100% ethanol was added into each cell lysate and the total 300 μllysates were transferred into one wells of “filter plate”. Filter platewas centrifuged at RCF of 10,000-15,000 g for 2 minutes. 300 μl WashSolution was then added into each well and the plate was centrifuged atRCF of 10,000-15,000 g for 2 minutes. For DNase treatment, 20 ul ofDNase mixture was added on top of each filter and the plate wasincubated for 15 minutes at room temperature. RNA rebinding wasperformed by washing filters with 200 μL of Rebinding Mix and 1 minutelater samples were centrifuged at RCF of 10,000-15,000 g for 2 minutes.Filter was washed then twice with 200 μl of Wash Solution andcentrifuged at RCF of 10,000-15,000 g for 2 minutes. A thirdcentrifugation of 2 minutes was then applied after the reservoir unitwas emptied and elution of the RNA was done into a clean culture plateby adding into the filters 50 μL of preheated (80° C.) Nuclease-freeWater.

cDNA Synthesis Using ABI High Capacity cDNA Reverse Transcription Kit(Applied Biosystems, Foster City, Calif., Cat #4368813):

A master mix of 2 μl 10× Buffer, 0.8 μl 25× dNTPs, 2 μl Random primers,1 μl Reverse Transcriptase, 1 μl RNase inhibitor and 3.2 μl of H2O perreaction were added into 10 μl total RNA. cDNA was generated using aBio-Rad C-1000 or S-1000 thermal cycler (Hercules, Calif.) through thefollowing steps: 25° C. 10 min, 37° C. 120 min, 85° C. 5 sec, 4° C.hold.

Real Time PCR:

2 μl of cDNA was added to a master mix of 1 μl GAPDH TaqMan Probe (HumanGAPD Endogenous Control VIC/MGB Probe, Primer Limited Applied BiosystemsCat #4326317E), 1 μl GNAQ TaqMan probe (Applied Biosystems cat #HS00387073_M1) and 10 μl TaqMan Universal PCR Master Mix (AppliedBiosystems Cat #4324018) per well in a MicroAmp Optical 96 well plate(Applied Biosystems cat #4326659). Real time PCR was done in an ABI7900HT Real Time PCR system (Applied Biosystems) using the ΔΔCt(RQ)assay. All reactions were done in triplicate.

Real time data were analyzed using the ΔΔCt method and normalized toassays performed from cells transfected with 10 nM BlockIT fluorescentOligo (Invitrogen Cat #2013) or 10 nM AD-1955 a duplex that targetsluciferase to calculate fold change.

Results

A total of 94 chemically modified siRNAs were screened. Single dosescreens were performed in A549 (lung carcinoma), A375 (malignantmelanoma) and uveal melanoma cell lines GNAQ^(mut), OMM1.3, and MEL202.Tables 8-14 show the results of the single-dose in vitro siRNA screen.

TABLE 8 A375 cells (0.1 nM) GNAQ dsRNA single dose in vitro screen A375cells (0.1 nM conc.) Sample Name Duplex Name % Target Remaining St. Deverror 26 AD-20057 44.52 4.74 21 AD-20052 49.83 4.55 20 AD-20051 51.946.86 38 AD-20069 53.68 5.80 60 AD-20092 54.34 5.94 66 AD-20099 56.065.86 14 AD-20045 56.35 5.74 91 AD-20193 57.53 3.82 68 AD-20101 58.444.72 23 AD-20054 60.08 6.13 89 AD-20103 60.82 5.02 16 AD-20047 61.665.59 53 AD-20085 61.99 8.52 56 AD-20088 62.09 7.48 81 AD-20116 63.847.76 19 AD-20050 64.39 8.36 78 AD-20113 64.84 7.95 8 AD-20039 65.29 9.2351 AD-20083 70.34 8.09 65 AD-20097 71.57 5.92 11 AD-20042 74.74 8.67 43AD-20074 74.87 6.70 47 AD-20079 75.39 6.12 24 AD-20055 77.24 36.15 58AD-20090 77.65 9.17 57 AD-20089 78.32 9.94 44 AD-20076 78.59 5.98 46AD-20078 79.00 5.54 64 AD-20096 80.39 5.96 48 AD-20080 80.66 10.31 84AD-20119 80.94 5.22 3 AD-20034 81.37 7.63 10 AD-20041 81.65 6.39 12AD-20043 81.65 11.97 6 AD-20037 81.79 11.99 59 AD-20091 81.79 9.24 13AD-20044 81.79 6.42 30 AD-20061 85.41 8.80 63 AD-20095 85.71 8.69 18AD-20049 85.71 10.26 75 AD-20110 86.60 13.52 52 AD-20084 87.81 11.94 69AD-20102 87.81 6.48 94 AD-20196 88.58 6.22 71 AD-20106 88.73 9.38 70AD-20104 89.35 14.88 35 AD-20066 89.81 7.73 54 AD-20086 89.97 12.64 45AD-20077 90.28 10.05 72 AD-20107 90.59 5.99 83 AD-20118 90.75 8.80 34AD-20065 91.54 12.41 62 AD-20094 92.02 10.71 74 AD-20109 92.82 11.79 79AD-20114 92.82 11.48 73 AD-20108 93.14 8.59 80 AD-20115 93.47 9.77 93AD-20195 93.63 7.82 55 AD-20087 93.95 15.29 76 AD-20111 93.95 14.29 92AD-20194 94.44 6.29 82 AD-20117 94.61 12.80 15 AD-20046 94.61 10.42 22AD-20053 94.93 16.04 77 AD-20112 95.10 12.23 29 AD-20060 95.10 11.08 67AD-20100 95.26 11.16 28 AD-20059 95.43 11.09 32 AD-20063 95.93 14.32 25AD-20056 96.09 12.23 90 96.26 9.50 95 AD-20105 96.76 10.01 9 AD-2004097.10 7.88 17 AD-20048 97.10 11.44 88 AD-20098 97.27 6.97 61 AD-2009397.27 12.46 39 AD-20070 97.43 9.70 7 AD-20038 97.60 11.22 87 97.94 8.3749 AD-20081 98.45 9.22 31 AD-20062 98.62 13.40 86 AD-20121 98.62 10.7150 AD-20082 98.79 12.83 41 AD-20072 98.97 9.54 42 AD-20073 99.48 9.92 85AD-20120 99.65 7.42 27 AD-20058 99.83 16.38 33 AD-20064 100.35 10.82 1AD-20032 101.40 9.56 37 AD-20068 101.57 9.44 4 AD-20035 102.99 15.49 2AD-20033 103.71 13.63 40 AD-20071 104.25 10.82 5 AD-20036 106.25 23.63

TABLE 9 A375 cells (1.0 nM) single dose GNAQ in vitro screen A375 cells(1 nM conc.) Sample Name Duplex Name % Target Remaining St. Dev error 26AD-20057 39.55 7.92 21 AD-20052 41.23 9.20 38 AD-20069 44.42 6.19 68AD-20101 45.04 6.93 20 AD-20051 45.11 8.89 14 AD-20045 45.98 7.80 19AD-20050 47.11 11.07 53 AD-20085 47.52 9.93 56 AD-20088 47.60 9.91 16AD-20047 48.35 8.35 66 AD-20099 48.44 8.52 78 AD-20113 48.69 8.81 81AD-20116 49.20 9.77 23 AD-20054 49.71 8.20 89 AD-20103 49.80 7.27 91AD-20193 51.29 8.73 65 AD-20097 52.27 9.60 60 AD-20092 52.46 6.04 51AD-20083 55.64 9.34 58 AD-20090 57.30 9.63 8 AD-20039 57.70 15.80 11AD-20042 58.51 9.24 43 AD-20074 59.43 9.18 24 AD-20055 59.53 13.24 47AD-20079 59.74 8.98 57 AD-20089 59.94 11.78 46 AD-20078 61.10 12.31 18AD-20049 61.31 8.08 30 AD-20061 63.14 11.19 6 AD-20037 63.91 10.65 10AD-20041 64.25 12.25 59 AD-20091 64.36 10.87 3 AD-20034 65.26 12.38 13AD-20044 65.26 10.78 64 AD-20096 66.51 10.16 44 AD-20076 66.86 8.87 93AD-20195 67.44 8.49 12 AD-20043 68.74 12.02 94 AD-20196 68.98 12.81 35AD-20066 69.70 10.38 54 AD-20086 70.79 13.09 45 AD-20077 71.04 10.55 84AD-20119 71.28 8.50 52 AD-20084 71.53 14.24 34 AD-20065 72.15 12.77 29AD-20060 74.44 11.33 48 AD-20080 74.83 9.32 63 AD-20095 75.09 12.36 75AD-20110 75.35 15.56 92 AD-20194 76.40 12.95 70 AD-20104 76.67 10.72 28AD-20059 78.41 12.71 74 AD-20109 78.55 15.25 15 AD-20046 78.69 12.82 55AD-20087 79.37 12.92 69 AD-20102 80.90 11.23 31 AD-20062 80.90 15.87 4AD-20035 81.18 19.43 83 AD-20118 82.45 19.97 49 AD-20081 82.60 15.95 67AD-20100 82.88 13.22 42 AD-20073 83.32 14.05 25 AD-20056 84.19 16.11 62AD-20094 84.48 13.94 41 AD-20072 84.92 12.80 9 AD-20040 85.21 15.48 71AD-20106 85.51 16.45 90 85.81 15.45 7 AD-20038 86.85 16.09 79 AD-2011487.76 16.56 33 AD-20064 88.07 20.64 80 AD-20115 88.07 17.42 2 AD-2003388.68 16.03 61 AD-20093 89.76 13.56 32 AD-20063 90.07 14.83 36 AD-2006790.23 9.73 77 AD-20112 90.54 15.45 86 AD-20121 91.49 20.81 95 AD-2010591.65 17.40 22 AD-20053 91.97 20.15 5 AD-20036 92.13 23.89 37 AD-2006892.77 14.46 39 AD-20070 93.09 16.90 27 AD-20058 93.09 17.29 17 AD-2004893.25 14.32 88 AD-20098 93.25 14.60 82 AD-20117 93.41 17.84 40 AD-2007194.39 15.66 50 AD-20082 94.88 17.58 87 95.71 15.99 1 AD-20032 96.7114.37 73 AD-20108 96.71 17.36 85 AD-20120 97.04 11.67 72 AD-20107 108.0512.36

TABLE 10 A549 cells (1.0 nM) single dose GNAQ in vitro screen A549 cells(1 nM conc.) Sample Name Duplex Name % Target Remaining St. Dev error 78AD-20113 13.33 2.98 53 AD-20085 15.79 3.53 81 AD-20116 16.44 3.68 21AD-20052 16.90 3.78 20 AD-20051 17.31 3.87 38 AD-20069 17.71 3.96 66AD-20099 17.77 3.98 19 AD-20050 18.11 4.05 64 AD-20096 18.17 4.07 26AD-20057 18.75 4.20 89 AD-20103 19.11 4.28 43 AD-20074 19.28 4.31 51AD-20083 19.41 4.34 68 AD-20101 19.61 4.39 14 AD-20045 20.06 4.49 8AD-20039 20.20 4.52 11 AD-20042 20.41 4.57 65 AD-20097 20.99 4.70 60AD-20092 21.02 4.70 56 AD-20088 22.53 5.04 44 AD-20076 22.57 5.05 58AD-20090 23.29 5.21 57 AD-20089 23.29 5.21 47 AD-20079 23.69 5.30 74AD-20109 23.86 5.34 16 AD-20047 24.02 5.38 63 AD-20095 24.36 5.45 59AD-20091 25.04 5.60 23 AD-20054 25.17 5.63 45 AD-20077 25.61 5.73 48AD-20080 25.84 5.78 91 AD-20193 28.92 6.47 13 AD-20044 29.83 6.68 6AD-20037 30.89 6.91 46 AD-20078 31.10 6.96 24 AD-20055 31.64 7.08 85AD-20120 31.70 7.09 18 AD-20049 33.74 7.55 84 AD-20119 34.75 7.77 3AD-20034 35.85 8.02 35 AD-20066 36.73 8.22 70 AD-20104 36.92 8.26 12AD-20043 38.62 8.64 54 AD-20086 38.96 8.72 15 AD-20046 39.98 8.95 34AD-20065 40.19 8.99 93 AD-20195 41.18 9.21 75 AD-20110 41.18 9.21 69AD-20102 41.68 9.33 52 AD-20084 42.19 9.44 30 AD-20061 44.29 9.91 94AD-20196 48.13 10.77 40 AD-20071 48.21 10.79 49 AD-20081 48.72 10.90 10AD-20041 48.80 10.92 36 AD-20067 48.97 10.96 29 AD-20060 50.79 11.36 31AD-20062 51.05 11.42 90 52.12 11.66 55 AD-20087 52.30 11.70 61 AD-2009352.85 11.83 2 AD-20033 53.50 11.97 25 AD-20056 55.77 12.48 4 AD-2003556.25 12.59 1 AD-20032 57.43 12.85 92 AD-20194 60.19 13.47 42 AD-2007361.03 13.65 5 AD-20036 61.45 13.75 28 AD-20059 61.99 13.87 50 AD-2008262.09 13.89 67 AD-20100 63.29 14.16 83 AD-20118 64.06 14.33 62 AD-2009464.17 14.36 27 AD-20058 64.95 14.53 7 AD-20038 69.26 15.50 79 AD-2011471.45 15.99 39 AD-20070 72.07 16.13 41 AD-20072 74.61 16.69 86 AD-2012174.61 16.69 33 AD-20064 75.39 16.87 9 AD-20040 80.11 17.92 72 AD-2010782.22 18.40 95 AD-20105 86.90 19.45 73 AD-20108 87.96 19.68 17 AD-2004889.04 19.92 88 AD-20098 90.13 20.17 77 AD-20112 90.44 20.24 80 AD-2011591.07 20.38 22 AD-20053 91.86 20.55 37 AD-20068 92.50 20.70 32 AD-2006392.66 20.73 76 AD-20111 92.82 20.77 71 AD-20106 92.98 20.80 82 AD-20117109.81 24.57 87 110.19 24.65

TABLE 11 OMM1.3 cells (10 nM) single dose GNAQ in vitro screen OMM1.3cells (10 nM conc.) Sample Name Duplex Name % Target Remaining St. Deverror 85 AD-20120 51.12 7.27 58 AD-20090 51.83 11.83 89 AD-20103 53.576.93 68 AD-20101 54.50 10.88 64 AD-20096 54.60 9.30 57 AD-20089 54.9810.87 53 AD-20085 55.07 11.92 38 AD-20069 55.55 10.05 59 AD-20091 55.9413.82 51 AD-20083 56.42 13.08 60 AD-20092 56.72 11.64 65 AD-20097 57.618.03 45 AD-20077 57.81 11.18 63 AD-20095 57.81 10.19 43 AD-20074 58.0111.58 91 AD-20193 58.11 10.38 26 AD-20057 58.21 10.36 20 AD-20051 58.417.60 24 AD-20055 58.92 9.65 66 AD-20099 59.74 10.83 44 AD-20076 59.7412.63 23 AD-20054 59.95 8.25 47 AD-20079 60.06 11.09 56 AD-20088 60.0612.78 61 AD-20093 60.06 13.48 41 AD-20072 60.37 12.49 13 AD-20044 61.1112.23 35 AD-20066 61.32 11.53 90 61.53 10.72 19 AD-20050 61.64 10.53 14AD-20045 61.85 7.21 15 AD-20046 61.96 10.96 21 AD-20052 62.07 7.36 34AD-20065 62.61 8.87 29 AD-20060 62.71 11.52 16 AD-20047 62.93 8.91 93AD-20195 63.26 10.94 69 AD-20102 63.59 7.49 54 AD-20086 64.25 16.58 50AD-20082 64.59 16.58 94 AD-20196 64.70 9.71 48 AD-20080 64.70 12.16 30AD-20061 64.81 9.02 A2 65.26 13.18 70 AD-20104 65.83 8.26 A3 66.41 11.4318 AD-20049 68.27 11.57 49 AD-20081 68.75 15.03 55 AD-20087 69.35 14.2531 AD-20062 69.71 10.58 52 AD-20084 71.42 17.10 A4 72.29 8.52 67AD-20100 73.68 15.34 27 AD-20058 74.19 12.01 36 AD-20067 74.32 17.93 33AD-20064 75.23 14.71 72 AD-20107 75.88 10.61 28 AD-20059 76.94 13.68 A176.94 14.61 71 AD-20106 77.08 12.79 25 AD-20056 79.11 12.52 8 AD-2003980.21 10.01 39 AD-20070 80.49 14.56 88 AD-20098 80.63 11.15 40 AD-2007180.77 16.38 62 AD-20094 81.75 16.23 86 AD-20121 84.49 9.13 17 AD-2004884.64 16.94 12 AD-20043 86.87 14.40 22 AD-20053 87.93 14.30 11 AD-2004288.23 13.27 37 AD-20068 91.66 17.18 32 AD-20063 91.98 14.78 87 94.5610.00 9 AD-20040 96.89 12.28 6 AD-20037 97.90 16.58 2 AD-20033 100.4817.62 3 AD-20034 100.83 12.65 1 AD-20032 105.84 19.01 7 AD-20038 114.6216.88 5 AD-20036 115.42 14.21 4 AD-20035 123.49 11.58 10 AD-20041 135.0565.85

TABLE 12 OMM1.3 cells (10 nM) single dose GNAQ in vitro screen OMM1.3(10 nM conc.) Sample Name Duplex Name % Target Remaining St. Dev error38 AD-20069 50.04 6.45 68 AD-20101 50.30 7.35 53 AD-20085 51.09 11.53 66AD-20099 51.45 8.97 64 AD-20096 51.72 8.35 43 AD-20074 53.17 6.93 21AD-20052 53.54 8.56 51 AD-20083 53.54 10.85 58 AD-20090 53.82 9.62 45AD-20077 54.29 8.36 26 AD-20057 54.76 12.81 56 AD-20088 54.86 12.18 65AD-20097 54.86 7.64 89 AD-20103 55.24 9.10 63 AD-20095 55.33 9.42 23AD-20054 55.53 7.94 19 AD-20050 55.53 8.95 57 AD-20089 55.82 10.84 91AD-20193 56.01 10.65 16 AD-20047 56.20 8.85 20 AD-20051 56.50 9.35 47AD-20079 56.50 7.15 15 AD-20046 56.69 7.92 44 AD-20076 57.09 8.01 59AD-20091 57.09 9.56 8 AD-20039 57.29 7.18 61 AD-20093 57.58 10.14 14AD-20045 57.78 10.19 85 AD-20120 57.78 9.98 54 AD-20086 57.88 9.77 11AD-20042 58.90 11.84 13 AD-20044 59.41 11.72 48 AD-20080 60.55 9.45 41AD-20072 60.87 6.66 A2 61.08 9.29 12 AD-20043 61.72 13.93 35 AD-2006661.72 11.27 6 AD-20037 61.72 9.69 69 AD-20102 61.93 10.90 34 AD-2006562.15 12.75 60 AD-20092 62.25 10.76 50 AD-20082 62.80 11.11 3 AD-2003463.12 7.93 10 AD-20041 63.89 9.55 18 AD-20049 64.00 9.43 30 AD-2006164.12 10.78 29 AD-20060 64.23 12.25 70 AD-20104 65.35 10.65 52 AD-2008467.54 14.15 55 AD-20087 67.77 12.89 90 67.77 10.53 24 AD-20055 68.3610.68 9 AD-20040 68.60 11.16 5 AD-20036 69.08 10.37 93 AD-20195 69.4410.54 2 AD-20033 70.04 12.33 31 AD-20062 71.02 11.73 49 AD-20081 71.0211.45 39 AD-20070 71.51 9.18 27 AD-20058 71.88 11.58 67 AD-20100 72.019.95 94 AD-20196 72.26 15.50 A1 72.89 8.79 33 AD-20064 73.39 13.32 A473.65 12.42 A3 74.55 14.37 25 AD-20056 74.81 12.70 28 AD-20059 74.9414.91 40 AD-20071 75.20 10.84 36 AD-20067 76.64 12.54 71 AD-20106 76.6410.67 4 AD-20035 76.91 10.25 22 AD-20053 78.80 15.37 86 AD-20121 79.4810.26 7 AD-20038 79.62 10.32 17 AD-20048 80.59 13.99 88 AD-20098 81.0112.34 72 AD-20107 82.00 16.19 62 AD-20094 82.43 14.56 32 AD-20063 84.6012.39 37 AD-20068 93.22 16.05 87 94.52 14.29 1 AD-20032 115.87 15.00

TABLE 13 UMEL 202 cells (10 nM) single dose GNAQ in vitro screen UMEL202 cells (10 nm Conc. Sample Name Duplex Name % Target Remaining St.Dev error 51 AD-20083 17.87 3.17 85 AD-20120 18.37 4.48 45 AD-2007718.76 5.42 68 AD-20101 18.82 3.16 26 AD-20057 19.42 3.43 64 AD-2009619.66 3.25 15 AD-20046 19.83 4.71 58 AD-20090 19.90 4.30 57 AD-2008920.74 4.31 53 AD-20085 21.55 4.68 89 AD-20103 22.15 4.48 63 AD-2009522.31 2.70 21 AD-20052 22.46 4.02 11 AD-20042 22.66 2.36 59 AD-2009122.78 4.06 20 AD-20051 22.86 3.46 38 AD-20069 23.34 5.47 16 AD-2004723.58 3.90 43 AD-20074 23.62 5.71 19 AD-20050 23.87 4.41 8 AD-2003923.91 2.96 14 AD-20045 24.33 4.08 47 AD-20079 25.10 5.85 50 AD-2008225.27 4.51 3 AD-20034 25.49 4.73 61 AD-20093 25.54 4.75 60 AD-2009225.76 4.91 56 AD-20088 25.94 3.59 66 AD-20099 26.03 4.28 65 AD-2009726.30 4.82 41 AD-20072 27.09 6.80 13 AD-20044 27.61 5.71 2 AD-2003327.70 3.68 91 AD-20193 27.90 4.64 29 AD-20060 27.99 5.33 44 AD-2007628.04 7.95 A2 28.29 6.73 54 AD-20086 28.78 5.43 69 AD-20102 29.18 4.7948 AD-20080 29.28 8.11 5 AD-20036 30.90 4.82 A3 30.95 6.03 18 AD-2004931.06 4.57 6 AD-20037 31.17 3.46 30 AD-20061 31.49 6.96 35 AD-2006631.71 39.01 34 AD-20065 34.05 7.29 90 34.11 5.07 94 AD-20196 34.17 6.4823 AD-20054 34.46 5.09 12 AD-20043 34.70 2.93 10 AD-20041 34.76 6.00 55AD-20087 36.55 8.21 31 AD-20062 36.81 7.00 49 AD-20081 37.06 7.58 25AD-20056 39.04 9.55 70 AD-20104 39.59 6.12 52 AD-20084 39.93 6.61 A440.42 7.46 93 AD-20195 41.99 7.10 40 AD-20071 42.28 8.86 27 AD-2005843.40 9.27 4 AD-20035 47.00 6.36 24 AD-20055 47.08 6.37 A1 48.65 10.76 9AD-20040 50.46 8.04 28 AD-20059 50.63 12.97 39 AD-20070 51.43 9.23 36AD-20067 52.42 10.11 33 AD-20064 52.78 10.02 17 AD-20048 54.36 8.74 88AD-20098 55.50 9.72 86 AD-20121 57.16 8.59 67 AD-20100 58.87 8.34 22AD-20053 65.32 10.91 62 AD-20094 68.10 10.87 7 AD-20038 72.48 9.86 37AD-20068 74.00 17.25 71 AD-20106 82.39 11.32 32 AD-20063 83.11 17.34 72AD-20107 89.39 11.20 87 99.18 18.11 1 AD-20032 119.33 18.54

TABLE 14 MEL 202 cells (10 nM) single dose GNAQ in vitro screen UMEL202cells (10 nM) Sample Name Duplex Name % Target Remaining St. Dev error85 AD-20120 16.28 1.84 26 AD-20057 18.41 3.50 68 AD-20101 18.73 3.64 45AD-20077 19.09 4.41 64 AD-20096 19.33 4.19 21 AD-20052 21.08 3.11 51AD-20083 21.22 4.27 58 AD-20090 22.36 4.62 63 AD-20095 22.55 3.04 20AD-20051 23.22 2.94 53 AD-20085 23.43 4.97 57 AD-20089 23.43 4.55 8AD-20039 24.00 3.73 89 AD-20103 24.25 5.69 15 AD-20046 24.30 3.82 38AD-20069 25.02 5.88 19 AD-20050 25.11 3.28 11 AD-20042 25.20 3.93 16AD-20047 25.20 3.98 59 AD-20091 25.41 5.04 43 AD-20074 25.50 6.00 61AD-20093 25.50 4.07 66 AD-20099 25.68 3.88 65 AD-20097 25.90 2.90 56AD-20088 25.95 4.67 47 AD-20079 26.31 5.00 41 AD-20072 26.96 5.21 69AD-20102 27.19 3.81 14 AD-20045 27.72 5.20 13 AD-20044 28.10 5.06 50AD-20082 28.25 5.67 54 AD-20086 28.35 4.75 60 AD-20092 28.84 4.72 29AD-20060 29.04 5.29 2 AD-20033 29.24 4.55 91 AD-20193 29.30 7.31 35AD-20066 29.40 6.42 3 AD-20034 29.45 5.51 A2 30.70 5.81 48 AD-2008030.86 7.14 44 AD-20076 31.07 7.63 12 AD-20043 31.29 7.00 30 AD-2006131.40 5.57 94 AD-20196 32.22 8.75 A3 32.73 7.68 18 AD-20049 33.36 6.21 5AD-20036 34.12 4.63 34 AD-20065 34.60 4.89 6 AD-20037 34.66 5.71 70AD-20104 35.32 5.17 23 AD-20054 35.39 5.08 90 36.19 8.71 10 AD-2004136.82 5.22 A4 36.89 11.72 93 AD-20195 37.73 9.95 31 AD-20062 37.93 7.8625 AD-20056 40.51 8.37 55 AD-20087 40.65 10.00 52 AD-20084 41.72 7.24 24AD-20055 43.26 6.08 49 AD-20081 43.34 13.40 27 AD-20058 45.57 7.25 A145.89 8.52 4 AD-20035 46.13 8.13 28 AD-20059 48.25 8.52 36 AD-2006748.84 13.01 40 AD-20071 48.93 9.64 88 AD-20098 50.30 12.25 33 AD-2006450.48 7.61 9 AD-20040 50.74 6.96 67 AD-20100 50.92 9.41 39 AD-2007053.36 14.44 17 AD-20048 53.45 6.78 22 AD-20053 66.61 12.90 86 AD-2012166.84 16.28 62 AD-20094 67.89 11.19 7 AD-20038 70.53 8.81 71 AD-2010681.44 14.31 32 AD-20063 83.29 12.02 72 AD-20107 85.04 14.05 87 100.2629.22 37 AD-20068 108.58 54.53 1 AD-20032 124.62 15.51

Duplexes with desirable levels of GNAQ inhibition were selected forfurther analysis of IC50 in A549 (lung carcinoma) MEL202 (GNAQ_(mut)uveal melanoma), and OMM1.3 cells (GNAQ_(mut) liver metastisis). Tables15-17 show the results of the IC50 experiments in A549, MEL202, andOMM1.3 cells. Dose response screen identified pM IC50s in lung carcinomacell line and GNAQmut uveal melanoma MEL202 and OMM1.3, includingduplexes AD-20057 and AD-20051.

TABLE 15 IC50 in A549 cells Rank Duplex Name IC50 in [nM] IC50 in [pM] 1AD-20057 0.0002 0.2 2 AD-20069 0.0026 2.6 3 AD-20051 0.0031 3.1 4AD-20052 0.0032 3.2 5 AD-20099 0.0033 3.3 6 AD-20045 0.0052 5.2 7AD-20193 0.0064 6.4 8 AD-20092 0.0094 9.4 9 AD-20116 0.0098 9.8 10AD-20039 0.0137 13.7 11 AD-20042 0.0172 17.2

TABLE 16 IC50 in MEL 202 cells Rank Duplex Name/(Sample Name) IC50 in[nM] 1 AD-20057 (26) 0.001 2 AD-20069 (38) 0.002 3 AD-20051 (20) 0.002 4AD-20052 (21) 0.003 5 AD-20045 (14) 0.003 6 AD-20193 (91) 0.003 7AD-20092 (60) 0.003 8 AD-20099 (66) 0.004 9 AD-20101 (68) 0.005 10AD-20116 (81) 0.006 11 AD-20039 (8)  0.006 12 AD-20103 (89) 0.007 13AD-20085 (53) 0.008 14 AD-20113 (78) 0.010 15 AD-20083 (51) 0.010 16AD-20096 (64) 0.010 17 AD-20042 (11) 0.011 18 AD-20090 (58) 0.023 19AD-20119 (84) 0.024 20 AD-20120 (85) 0.037 21 AD-20109 (74) 0.047 22AD-20077 (45) 0.084

TABLE 17 IC50 in OMM1.3 cells Rank Duplex Name (Sample) IC50 in [nM] 1AD-20057 (26) 0.0043 2 AD-20069 (38) 0.0115 3 AD-20052 (21) 0.0183 4AD-20051 (20) 0.0197 5 AD-20099 (66) 0.0270 6 AD-20092 (60) 0.0280 7AD-20193 (91) 0.0335 8 AD-20101 (68) 0.0531 9 AD-20045 (14) 0.0538 10AD-20113 (78) 0.0625 11 AD-20039 (8)  0.0693 12 AD-20103 (89) 0.0820 13AD-20085 (53) 0.0842 14 AD-20116 (81) 0.1280 15 AD-20083 (51) 0.1653 16AD-20042 (11) 0.2470 17 AD-20090 (58) 0.2593 18 AD-20096 (64) 0.3006 19AD-20120 (85) 0.6189 20 AD-20119 (84) 1.2276 21 AD-20109 (74) 1.2558 22AD-20077 (45) 2.0044

Example 4 In Vitro Dose Response

For in vitro dose response experiments, cells expressing GNAQ wereutilized. Some exemplary cell lines expressing GNAQ include, but are notlimited to, human melanoma cell lines OMM1.3 and Mel 202 and MEL-285.

The dsRNAs were screened for in vitro inhibition of the target gene at 1nM, 0.1 nM, 0.01 nM, and 0.001 nM. Tissue culture cells were transfectedwith the dsRNA. Target gene mRNA levels were assayed using qPCR (realtime PCR).

Cell Culture and Transfection

For knockdown, OMM-1.3, MEL-202 and MEL-285 were grown to nearconfluence at 37° C. in an atmosphere of 5% CO₂ in RPMI (Invitrogen)supplemented with 10% FBS, streptomycin, and glutamine (ATCC) beforebeing released from the plate by trypsinization. Reverse transfectionwas carried out by adding 5 μl of Opti-MEM to 5 μl of siRNA duplexes perwell into a 96-well plate along with 10 μl of Opti-MEM plus 0.2 μl ofLipofectamine RNAiMax per well (Invitrogen, Carlsbad Calif. cat#13778-150) and incubated at room temperature for 15 minutes. 80 μl ofcomplete growth media without antibiotic containing 2.0×10⁴ OMM-1.3,MEL-202 or MEL-285 cells were then added. Cells were incubated for 24hours prior to RNA purification. Experiments were performed at 1, 0.1,0.01 and 0.001 nM final duplex concentration.

Total RNA Isolation Using MagMAX-96 Total RNA Isolation Kit (AppliedBiosystem, Foster City Calif., Part #: AM1830):

Cells were harvested and lysed in 140 μl of Lysis/Binding Solution thenmixed for 1 minute at 850 rpm using and Eppendorf Thermomixer (themixing speed was the same throughout the process). Twenty micro litersof magnetic beads and Lysis/Binding Enhancer mixture were added intocell-lysate and mixed for 5 minutes. Magnetic beads were captured usingmagnetic stand and the supernatant was removed without disturbing thebeads. After removing supernatant, magnetic beads were washed with WashSolution 1 (isopropanol added) and mixed for 1 minute. Beads werecapture again and supernatant removed. Beads were then washed with 150μl Wash Solution 2 (Ethanol added), captured and supernatant wasremoved. 50 μl of DNase mixture (MagMax turbo DNase Buffer and TurboDNase) was then added to the beads and they were mixed for 10 to 15minutes. After mixing, 100 μl of RNA Rebinding Solution was added andmixed for 3 minutes. Supernatant was removed and magnetic beads werewashed again with 150 μl Wash Solution 2 and mixed for 1 minute andsupernatant was removed completely. The magnetic beads were mixed for 2minutes to dry before RNA was eluted with 50 μl of water.

cDNA Synthesis Using ABI High Capacity cDNA Reverse Transcription Kit(Applied Biosystems, Foster City, Calif., Cat #4368813):

A master mix of 2 μl 10× Buffer, 0.8μl 25× dNTPs, 2 μl Random primers, 1μl Reverse Transcriptase, 1 μl RNase inhibitor and 3.2 μl of H₂O perreaction were added into 10 μl total RNA. cDNA was generated using aBio-Rad C-1000 or S-1000 thermal cycler (Hercules, Calif.) through thefollowing steps: 25° C. 10 min, 37° C. 120 min, 85° C. 5 sec, 4° C.hold.

Real Time PCR:

2 μl of cDNA were added to a master mix containing 0.5 μl GAPDH TaqManProbe

(Applied Biosystems Cat #4326317E), 0.5 μl GNAQ TaqMan probe (AppliedBiosystems cat # Hs00387073_ml) and 5 μl Roche Probes Master Mix (RocheCat #04887301001) per well in a LightCycler 480 384 well plate (Rochecat #0472974001). Real time PCR was done in a LightCycler 480 Real TimePCR machine (Roche). Each duplex was tested in two independenttransfections and each transfections was assayed in duplicate.

Real time data were analyzed using the ΔΔCt method. Each sample wasnormalized to GAPDH expression and knockdown was assessed relative tocells transfected with the non-targeting duplex AD-1955.

The data are presented in Table 18a. Data are expressed as the fractionof message remaining relative to cells targeted with AD-1955. Thecalculated IC₅₀s are presented in Table 18b.

TABLE 18a In vitro dose response in 3 cell lines Duplex name 1 nM 0.1 nM0.01 nM 0.001 nM OMM-1.3 AD-20039 0.38 0.46 0.74 0.73 AD-20045 0.42 0.520.60 0.79 AD-20051 0.34 0.46 0.63 1.18 AD-20052 0.36 0.37 0.53 0.61AD-20057 0.32 0.36 0.43 0.59 AD-20063 0.63 0.69 0.99 0.74 AD-20069 0.370.35 0.43 0.69 AD-20092 0.42 0.51 0.71 0.75 AD-20099 0.35 0.46 0.52 0.63AD-20101 0.39 0.57 0.60 0.69 AD-20111 0.64 0.68 0.65 0.70 AD-20113 0.370.51 0.71 0.92 AD-20116 0.56 0.58 0.66 0.75 AD-20193 0.45 0.50 0.64 0.75AD-1955  1.12 1.17 0.83 0.92 MEL-202 AD-20039 0.35 0.44 0.63 0.83AD-20045 0.30 0.36 0.53 0.55 AD-20051 0.22 0.37 0.67 0.88 AD-20052 0.330.39 0.66 0.85 AD-20057 0.28 0.29 0.39 0.77 AD-20063 0.93 0.87 0.95 0.97AD-20069 0.35 0.39 0.39 0.75 AD-20092 0.37 0.49 0.93 0.98 AD-20099 0.280.33 0.61 0.96 AD-20101 0.38 0.46 0.83 0.92 AD-20111 0.67 0.81 0.91 0.98AD-20113 0.31 0.48 0.82 0.99 AD-20116 0.33 0.34 0.72 0.92 AD-20193 0.320.44 0.65 0.87 AD-1955  1.11 0.85 1.11 0.95 MEL-285 AD-20039 0.29 0.470.95 1.09 AD-20045 0.39 0.42 0.69 0.86 AD-20051 0.34 0.34 0.73 0.90AD-20052 0.30 0.53 1.17 1.22 AD-20057 0.37 0.34 0.54 0.86 AD-20063 0.991.05 1.52 1.37 AD-20069 0.27 0.33 0.55 0.80 AD-20092 0.39 0.58 0.78 0.82AD-20099 0.28 0.40 0.92 1.10 AD-20101 0.35 0.57 0.82 1.05 AD-20111 0.750.79 0.78 0.73 AD-20113 0.32 0.53 0.92 1.18 AD-20116 0.55 0.51 1.17 0.91AD-20193 0.42 0.47 0.79 0.95 AD-1955  0.93 1.01 0.93 1.15

TABLE 18b IC₅₀ (pM) in 3 cell lines duplex number MEL202 OMM1.3 A549AD-20057 0.7 4.3 0.2 AD-20069 1.8 11.5 2.6 AD-20051 2.5 19.7 3.1AD-20052 2.6 18.3 3.2 AD-20045 2.8 53.8 5.2 AD-20193 3.2 33.5 6.4AD-20092 3.5 28 9.4 AD-20099 3.6 27 3.3 AD-20101 4.9 53.1 AD-20116 5.5128 9.8 AD-20113 9.5 62.5 AD-20039 6.1 69.3 13.7

Example 5 Immunostimulatory Assays: Screening siRNA Sequences forImmunostimulatory Ability

Twelve siRNA candidates were tested for induction of cytokinesassociated with immunostimulation (TNF-alpha and IFN-alpha).

Human PBMC were isolated from whole blood from healthy donors (ResearchBlood Components, Inc., Boston, Mass.) by a standard Ficoll-Hypaquedensity gradient centrifugation technique. PBMC (1×10⁵/well/100 μL) wereseeded in 96-well flat bottom plates and cultured in RPMI 1640GlutaMax-1 medium (Invitrogen) supplemented with 10% heat-inactivatedfetal bovine serum (Omega Scientific) and 1% antibiotic/antimycotic(Invitrogen).

GNAC siRNAs was transfected into PBMC using N-[1-(2,3-Dioleoyloxy)propyl]-N,N,N-trimethylammonium methylsulfate (DOTAP;Roche). The DOTAP was first diluted in Opti-MEM Reduced Serum medium(Invitrogen) for 5 minutes before mixing with an equal volume ofOpti-MEM containing the siRNA. siRNA/DOTAP complexes were incubated for10-15 minutes at room temperature and subsequently added to PBMC (50μL/well) which were then cultured at 37° C. 5% CO₂. siRNAs were used ata final concentration of 133 nM. The ratio of RNA to transfectionreagent was 16.5 pmoles per μL of DOTAP. Transfections were conducted inquadruplicate in all experiments and were performed within two hours ofcell plating. Culture supernatants were collected after 20-24 h andassayed for IFN-α and TNF-α by ELISA.

Cytokines were detected and quantified in culture supernatants with acommercially available ELISA kit for IFN-α (BMS216INST) and TNF-α(BMS223INST) from Bender MedSystems (Vienna, Austria).

Results

The data in Table 19 are presented as a percentage to a AD-5048stimulated cytokine response. AD-5048 (positive control) corresponds toa sequence that targets human Apolipoprotein B (Soutschek et al., 2004)and elicits both an IFN-α and TNF-α. FIG. 1 and FIG. 2 shows thecytokine induction following transfection with siRNAs.

None of the siRNAs tested demonstrated significant expression of IFN-αand TNF-α in Human PBMCs compared to AD-5048. In particular, AD-20051and AD-20057 were found to be non immunostimulatory in HuPBMC assay.

TABLE 19 Immunostimulatory activity Duplex name % IFN-α/AD-5048 %TNF-α/AD-5048 AD-20039 0 0 AD-20045 0 0 AD-20051 0 0 AD-20052 0 0AD-20057 0 0 AD-20069 0 0 AD-20092 0 0 AD-20099 0 0 AD-20101 0 0AD-20113 0 0 AD-20116 0 0 AD-20193 0 0

Example 6 In Vitro Cell Viability

A set of dsRNAs were screened for effects on in vitro cell viability.Tissue culture cells were transfected with the dsRNA and viability wasassayed by staining with CellTiterBLue and microscopic evaluation.

Cell Culture and Transfection

For viability, OMM-1.3, MEL-202 and MEL-285 cells were grown to nearconfluence at 37° C. in an atmosphere of 5% CO₂ in RPMI, (Invitrogen)supplemented with 10% FBS, Penn/streptomycin, and glutamine (ATCC)before being released from the plate by trypsinization. Reversetransfection was carried out by adding 5 μl of Opti-MEM to 5 μl of siRNAduplexes per well into a 96-well plate along with 10 μl of Opti-MEM plus0.2 μl of Lipofectamine RNAiMax per well (Invitrogen, Carlsbad Calif.cat #13778-150) and incubated at room temperature for 15 minutes. 80 μlof complete growth media without antibiotic containing 1.0×10³ OMM-1.3,MEL-202 or MEL-285 cells were then added. Cells were incubated for 3, 5or 7 days prior to viability assays. Experiments were performed at 1,0.1, 0.01 and 0.001 nM final duplex concentration. All transfectionswere done in triplicate. The siRNAs PLK, and AD-19200 were included aspositive controls (result in loss of viability) and AD-1955 was includedas a negative control and was used for data normalization.

Cell Viability Assay

For viability assays, 20 μl of CellTiterBlue (Promega, Cat# G8080) wasadded and mixed into each well of the culture plate 3, 5 or 7 days aftertransfection with an siRNAs at 1, 0.1, 0.01 or 0.001 nM finalconcentration. The plates, containing transfected, cultured cells, mediaand CellTiterBlue were incubated for 1.5 hours and then read on aSpectraMax M5 plate reader (Molecular Devices) at 560 nm (excitation)and 590 nm (emission).

To measure viability, three replicate wells were averaged and subtractedfrom background (wells containing media and CellTiterBlue, but nocells). Viability is expressed as a normalized value in which cellstransfected with GNAQ specific siRNAs or other controls are compared tocells transfected with AD-1955, a non-targeting duplex, cultured underthe same conditions.

Results

The results are shown in Table 20. Graphical summaries of the resultscomparing viability at 3, 5, and 7 days in a single cell line aftertreatment with each of the duplexes at a single concentration are shownin FIG. 3, FIG. 4, FIG. 5, and FIG. 6.

The results show decreased cell viability in vitro following GNAQknockdown that was specific for GNAQ mutant cell lines (e.g., OMM1.3,MEL202), but not GNAQ wild-type (e.g., MEL285) cell lines. In particularthese results were shown for duplexes AD-20057, AD-20051, AD-20069, andAD-20093 as illustrated by the graphs in FIG. 7 and FIG. 8.

TABLE 20 Cell viability after treatment with siRNA Day 3 Day 5 Day 7Conc. (in nM) 1 nM 0.1 nM 0.01 nM 0.001 nM 1 nM 0.1 nM 0.01 nM 0.001 nM1 nM 0.1 nM 0.01 nM 0.001 nM OMM-1.3 AD-20039 0.95 0.96 1.18 1.27 0.460.48 0.79 0.94 0.25 0.52 1.18 1.32 AD-20045 0.99 0.94 1.07 1.20 0.530.53 0.70 1.03 0.42 0.44 0.71 1.16 AD-20051 0.78 0.90 1.01 0.63 0.350.42 0.65 0.94 0.23 0.35 0.74 1.15 AD-20052 0.82 0.90 1.17 1.38 0.410.47 0.79 1.02 0.31 0.45 1.06 1.24 AD-20057 0.86 0.88 0.90 1.31 0.360.39 0.49 0.83 0.22 0.31 0.55 1.03 AD-20063 1.26 1.26 1.10 0.53 1.271.06 1.04 0.93 1.11 0.95 1.00 1.06 AD-20069 0.79 0.72 0.96 1.16 0.350.39 0.46 0.86 0.17 0.21 0.58 0.89 AD-20092 0.68 0.93 1.11 1.15 0.360.58 0.85 0.91 0.27 0.63 1.08 0.96 AD-20099 0.51 0.72 0.95 1.07 0.180.37 0.58 0.88 0.08 0.22 0.56 0.86 AD-20101 0.72 0.76 1.34 1.53 0.330.40 0.78 1.00 0.18 0.39 0.98 0.95 AD-20111 1.25 1.15 1.30 1.32 0.891.10 0.87 1.00 0.93 0.95 0.98 0.95 AD-20113 0.56 0.73 1.02 1.03 0.220.44 0.75 0.80 0.12 0.35 0.88 0.82 AD-20116 0.82 1.23 1.64 1.88 0.410.70 0.92 0.98 0.17 0.51 0.75 0.73 AD-20193 1.22 0.84 1.31 1.67 0.460.53 0.66 0.80 0.16 0.30 0.48 0.74 AD-12115 0.60 0.65 1.03 1.00 0.190.26 0.62 0.93 0.08 0.26 0.63 0.61 PLK 0.47 0.80 0.65 1.67 0.12 0.540.74 1.00 0.06 0.64 0.88 0.78 AD-19200 0.62 0.85 0.72 1.55 0.64 0.780.92 0.81 0.64 0.83 0.81 0.87 AD-1955 1.00 1.00 1.00 1.00 1.00 1.00 1.001.00 1.00 1.00 1.00 1.00 MEL-202 AD-20039 1.21 0.98 1.02 0.93 0.72 0.560.79 1.00 0.78 0.66 0.75 0.88 AD-20045 0.95 0.90 0.95 0.85 0.47 0.380.61 0.92 0.57 0.36 0.47 0.80 AD-20051 0.70 0.78 0.80 0.38 0.62 0.360.77 0.82 0.82 0.42 0.62 0.80 AD-20052 0.98 1.02 1.06 0.93 0.46 0.420.67 1.02 0.33 0.55 0.95 1.03 AD-20057 0.61 0.91 0.86 0.85 0.30 0.270.47 0.86 0.31 0.34 0.53 0.88 AD-20063 0.91 1.00 1.02 0.37 0.74 0.811.01 0.91 1.54 1.32 1.08 1.04 AD-20069 0.77 1.03 0.91 1.00 0.28 0.440.43 0.64 0.34 0.37 0.49 0.79 AD-20092 0.87 0.88 0.95 0.87 0.25 0.440.70 0.80 0.26 0.73 0.97 1.11 AD-20099 0.80 0.69 0.75 0.41 0.24 0.360.53 0.68 0.13 0.26 0.66 1.16 AD-20101 0.72 0.92 0.80 0.87 0.16 0.480.57 0.73 0.17 0.51 1.21 0.92 AD-20111 1.18 0.90 0.75 0.84 0.67 0.830.74 0.80 1.30 1.37 1.25 1.03 AD-20113 0.63 0.55 0.74 0.36 0.18 0.310.66 0.68 0.15 0.37 1.00 1.05 AD-20116 0.42 0.59 0.62 0.93 0.41 0.510.73 0.90 0.29 0.38 0.71 0.77 AD-20193 0.39 0.53 0.53 0.94 0.36 0.490.70 0.76 0.29 0.32 0.59 0.80 AD-12115 0.22 0.22 0.30 0.50 0.09 0.120.42 0.78 0.03 0.04 0.08 0.68 PLK 0.23 0.27 0.37 0.63 0.10 0.16 0.560.65 0.03 0.07 0.97 1.19 AD-19200 0.37 0.52 0.49 0.56 0.29 0.75 0.760.74 0.44 1.15 1.04 0.85 AD-1955 1 1 1 1 1 1 1 1 1 1 1 1 MEL-285AD-20039 0.58 1.37 1.23 1.23 1.07 1.25 1.13 1.09 0.82 1.06 0.96 0.93AD-20045 1.16 1.31 1.15 1.05 1.10 1.10 1.12 1.24 0.84 0.85 0.97 0.90AD-20051 1.14 1.20 0.97 0.98 1.27 1.16 1.06 1.03 0.84 0.99 0.89 0.97AD-20052 0.63 1.40 1.26 1.04 1.03 1.40 1.42 1.21 0.92 1.02 1.13 0.98AD-20057 1.14 1.17 1.20 1.04 1.00 1.04 1.26 1.35 0.98 0.98 1.03 1.00AD-20063 1.10 1.14 0.88 0.89 1.33 1.14 0.94 0.95 1.16 1.05 0.87 0.91AD-20069 0.46 1.09 1.08 0.96 1.17 1.20 1.03 1.04 1.29 1.17 1.01 0.99AD-20092 1.02 1.14 1.15 0.96 1.02 1.11 1.03 1.05 1.06 1.11 1.04 1.02AD-20099 0.89 1.10 0.95 0.95 0.48 0.92 0.96 1.00 0.54 0.91 0.89 1.05AD-20101 0.70 1.16 1.12 1.04 0.47 1.12 1.41 1.42 0.66 1.01 1.22 1.03AD-20111 1.12 1.05 1.13 1.01 1.21 1.49 1.30 1.29 1.04 1.18 1.04 1.03AD-20113 0.81 0.97 1.02 1.03 0.41 0.85 0.81 0.97 0.31 0.76 0.85 1.01AD-20116 0.50 0.86 1.07 1.01 1.03 0.98 1.03 1.01 0.99 0.91 1.01 0.94AD-20193 0.91 0.88 1.03 0.94 0.58 0.86 1.25 1.24 0.72 0.80 1.09 1.03AD-12115 0.34 0.35 0.81 0.43 0.10 0.12 0.54 1.02 0.07 0.12 0.82 1.00 PLK0.23 0.65 0.46 0.97 0.31 0.54 1.40 1.31 0.18 0.72 1.38 1.17 AD-192000.53 0.81 0.68 0.94 0.53 0.77 1.22 1.32 0.46 0.97 1.22 1.15 AD-1955 1.001.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00

Example 7 In Vivo Efficacy Studies

The dsRNAs are screened for in vivo inhibition of the target gene inmice. Mice are injected with varying amounts of the dsRNA. Target geneprotein levels are assayed using, e.g., mouse plasma and an ELISA with atarget gene specific antibody. Target gene mRNA levels are assayedusing, e.g., mouse liver and branched DNA assays. The lead candidatesare dsRNA that reduce levels of the target gene protein and/or mRNA in adose-dependent manner.

Regimen for Treatment of Mice with dsRNA

A single-dose IV bolus efficacy study is designed for each dsRNA to betested: dose level, dosing days, formulation, and number of animals.Mice are intravenously (i.v.)

administered target gene specific dsRNA, control dsRNA) or PBSsystemically and/or subcutaneously in a range of concentrations, e.g.,1.0 mg/kg, 3.0 mg/kg, or 6.0 mg/kg.

Mice are observed for forty-hours then anesthetized with 200 μl ofketamine, and are exsanguinated by severing the right caudal artery.Whole blood is isolated and placed into EDTA plasma separator tubes andcentrifuged at 3000 rpm for 10 minutes. Plasma is isolated and stored at80° C. until assaying. Liver tissue is collected, flash-frozen andstored at −80° C. until processing.

Efficacy of treatment is evaluated by methods including (i) measurementof protein in plasma at prebleed and at 48 hours post-dose, (ii)measurement of mRNA in liver at 48 hours post-dose, and (iii) efficacyin modulation of target gene specific phenotype, e.g., anti-tumoractivity.

Assay of Target Gene Protein in Mouse Plasma

Target plasma levels are assayed by ELISA utilizing the commerciallyavailable anti GNAQ antibodies, for example G alpha q (K-17) or G alphaq (E-17) (Santa Cruz Biotechnology Inc. Santa Cruz, Calif., USA, cat#SC-26791 and cat # SC-393), according to manufacturer's guidelines.

Assay of Target Gene mRNA Levels in Mouse Liver

Target gene mRNA levels are assayed utilizing the Branched DNA assaysQuantigene 2.0 (Panomics cat #: QS0011). Briefly, mouse liver samplesare ground and tissue lysates are prepared. Liver lysis Mixture (amixture of 1 volume of lysis mixture, 2 volume of nuclease-free waterand 10 ul of Proteinase-K/ml for a final concentration of 20 mg/ml.) isincubated at 65° C. for 35 minutes. 20 μl of Working Probe Set (targetprobe for detection of target gene and GAPDH probe for endogenouscontrol) and 80 ul of tissue-lysate are then added into the CapturePlate. Capture Plates are incubated at 55° C.±1° C. (aprx. 16-20 hrs).The next day, the Capture Plate are washed 3 times with 1× Wash Buffer(nuclease-free water, Buffer Component 1 and Wash Buffer Component 2),then dried by centrifuging for 1 minute at 240 g. 100 ul of pre-AmpliferWorking Reagent is added into the Capture Plate, which is sealed withaluminum foil and incubated for 1 hour at 55° C.±1° C. Following 1 hourincubation, the wash step is repeated, then 100 μl of Amplifier WorkingReagent is added. After 1 hour, the wash and dry steps are repeated, and100 μl of Label Probe is added. Capture plates are incubated 50° C.±1°C. for 1 hour. The plate is then washed with 1× Wash Buffer, dried and100 μl Substrate is added into the Capture Plate. Capture Plates areread using the SpectraMax Luminometer following a 5 to 15 minuteincubation. bDNA data are analyzed by subtracting the average backgroundfrom each triplicate sample, averaging the triplicate GAPDH (controlprobe) and target gene probe (experimental probe) then taking the ratio:(experimental probe-background)/(control probe-background).

GNAQ Materials and Methods

The GNAQ specific dsRNA are formulated in lipid particles (SNALP) asdescribe herein and administered systemically or subcutaneously to micewith GNAQ-mutant human uveal melanoma cell tumors implanted in the liverto assess in vivo target knockdown and antitumor activity. The dsRNAduplexes with positive results are selected for further studies todevelop a Phase I/II trial in patients with GNAQ-mutant uveal melanomametastatic to liver.

Example 8 Inhibition of GNAQ in Humans

A human subject is treated with a dsRNA targeted to a GNAQ gene toinhibit expression of the GNAQ gene to treat a condition.

A subject in need of treatment is selected or identified. The subjectcan have uveal melanoma, cutaneous melanoma, Blue nevi, Nevi of Ota, aneuroendocrine tumor, or a small lung tumor.

The identification of the subject can occur in a clinical setting, orelsewhere, e.g., in the subject's home through the subject's own use ofa self-testing kit.

At time zero, a suitable first dose of an anti- GNAQ siRNA isadministered to the subject. The dsRNA is formulated as describedherein. After a period of time following the first dose, e.g., 7 days,14 days, and 21 days, the subject's condition is evaluated, e.g., bymeasuring tumor growth. This measurement can be accompanied by ameasurement of GNAQ expression in said subject, and/or the products ofthe successful siRNA-targeting of GNAQ mRNA. Other relevant criteria canalso be measured. The number and strength of doses are adjustedaccording to the subject's needs.

After treatment, the subject's tumor growth rate is lowered relative tothe rate existing prior to the treatment, or relative to the ratemeasured in a similarly afflicted but untreated subject.

Example 9 GNAQ mRNA Sequences

Human GNAQ mRNA NM_002072.2 (SEQ ID NO: 1761) 1agggggtgcc ggcggggctg cagcggaggc actttggaag aatgactctg gagtccatca 61tggcgtgctg cctgagcgag gaggccaagg aagcccggcg gatcaacgac gagatcgagc 121ggcagctccg cagggacaag cgggacgccc gccgggagct caagctgctg ctgctcggga 181caggagagag tggcaagagt acgtttatca agcagatgag aatcatccat gggtcaggat 241actctgatga agataaaagg ggcttcacca agctggtgta tcagaacatc ttcacggcca 301tgcaggccat gatcagagcc atggacacac tcaagatccc atacaagtat gagcacaata 361aggctcatgc acaattagtt cgagaagttg atgtggagaa ggtgtctgct tttgagaatc 421catatgtaga tgcaataaag agtttatgga atgatcctgg aatccaggaa tgctatgata 481gacgacgaga atatcaatta tctgactcta ccaaatacta tcttaatgac ttggaccgcg 541tagctgaccc tgcctacctg cctacgcaac aagatgtgct tagagttcga gtccccacca 601cagggatcat cgaatacccc tttgacttac aaagtgtcat tttcagaatg gtcgatgtag 661ggggccaaag gtcagagaga agaaaatgga tacactgctt tgaaaatgtc acctctatca 721tgtttctagt agcgcttagt gaatatgatc aagttctcgt ggagtcagac aatgagaacc 781gaatggagga aagcaaggct ctctttagaa caattatcac atacccctgg ttccagaact 841cctcggttat tctgttctta aacaagaaag atcttctaga ggagaaaatc atgtattccc 901atctagtcga ctacttccca gaatatgatg gaccccagag agatgcccag gcagcccgag 961aattcattct gaagatgttc gtggacctga acccagacag tgacaaaatt atctactccc 1021acttcacgtg cgccacagac accgagaata tccgctttgt ctttgctgcc gtcaaggaca 1081ccatcctcca gttgaacctg aaggagtaca atctggtcta attgtgcctc ctagacaccc 1141gccctgccct tccctggtgg gctattgaag atacacaaga gggactgtat ttctgtggaa 1201aacaatttgc ataatactaa tttattgccg tcctggactc tgtgtgagcg tgtccacaga 1261gtttgtagta aatattatga ttttatttaa actattcaga ggaaaaacag aggatgctga 1321agtacagtcc cagcacattt cctctctatc ttttttttag gcaaaacctt gtgactcagt 1381gtattttaaa ttctcagtca tgcactcaca aagataagac ttgtttcttt ctgtctctct 1441ctctttttct tttctatgga gcaaaacaaa gctgatttcc cttttttctt cccccgctaa 1501ttcatacctc cctcctgatg tttttcccag gttacaatgg cctttatcct agttccattc 1561ttggtcaagt ttttctctca aatgatacag tcaggacaca tcgttcgatt taagccatca 1621tcagcttaat ttaagtttgt agtttttgct gaaggattat atgtattaat acttacggtt 1681ttaaatgtgt tgctttggat acacacatag tttctttttt aatagaatat actgtcttgt 1741ctcactttgg actgggacag tggatgccca tctaaaagtt aagtgtcatt tcttttagat 1801gtttaccttc agccatagct tgattgctca gagaaatatg cagaaggcag gatcaaagac 1861acacaggagt cctttctttt gaaatgccac gtgccattgt ctttcctccc ttctttgctt 1921ctttttctta ccctctcttt caattgcaga tgccaaaaaa gatgccaaca gacactacat 1981taccctaatg gctgctaccc agaacctttt tataggttgt tcttaatttt tttgttgttg 2041ttgttcaagc ttttcctttc ttttttttct tagtgtttgg gccacgattt taaaatgact 2101tttattatgg gtatgtgttg ccaaagctgg ctttttgtca aataaaatga atacgaactt 2161aaaaaataaa aaaaaaaaaa aaaaaaaa Rat GNAQ mRNA NM_031036 (SEQ ID NO: 1762)1 atgactctgg agtccatcat ggcgtgctgc ctgagcgagg aggccaagga agcccggagg 61atcaacgacg agatcgagcg gcagctgcgc agggacaagc gcgacgcccg ccgggagctc 121aagctgctgc tgctggggac aggggagagt ggcaagagta ccttcattaa gcagatgagg 181atcatccacg ggtcggggta ctctgatgaa gacaagaggg gctttaccaa actggtgtat 241cagaacatct ttacagccat gcaggccatg gtcagagcta tggacactct caagatccca 301tacaagtatg aacacaataa ggctcatgca caattggttc gagaggttga tgtggagaag 361gtgtctgctt ttgagaatcc atatgtagac gcaataaaga gcttgtggaa tgatcctgga 421atccaggaat gctacgatag acggcgagaa tatcagctat ctgactctac caaatactat 481ctgaacgact tggaccgtgt ggctgaccct tcctatctgc ctacacaaca agatgtgctt 541agagttcgag tccccaccac agggatcatt gagtacccct tcgacttaca gagtgtcatc 601ttcagaatgg tcgatgtagg aggccaaagg tcagagagaa gaaaatggat acactgcttt 661gaaaacgtca cctcgatcat gtttctggta gcgcttagcg aatacgatca agttcttgtg 721gagtcagaca atgagaaccg aatggaggag agcaaagcac tctttagaac cattatcaca 781tatccctggt tccagaactc ctctgttatt ctgttcttaa acaagaaaga tcttctagag 841gagaaaatta tgtattccca cctagtcgac tacttcccag aatatgatgg accccagaga 901gatgcccagg cagcacgaga attcatcctg aagatgttcg tggacctgaa ccccgacagt 961gacaaaatca tctactcgca cttcacgtgt gccacagaca cggagaacat ccgcttcgtg 1021tttgctgctg tcaaggacac catcctgcag ctgaacctga aggagtacaa tctggtctaa

We claim:
 1. An isolated double-stranded ribonucleic acid (dsRNA) forinhibiting expression of a G-alpha q subunit (GNAQ) of a heterotrimericG gene, comprising a sense strand and an antisense strand and theantisense strand is complementary to at least 15 contiguous nucleotidesof SEQ ID NO:
 1395. 2. The dsRNA of claim 1, comprising a modificationthat causes the dsRNA to have increased stability in a biologicalsample.
 3. The dsRNA of claim 1, comprising at least one modifiednucleotide.
 4. The dsRNA of claim 3, wherein said modified nucleotide isselected from the group of: a 2′-O-methyl modified nucleotide, anucleotide comprising a 5′-phosphorothioate group, a terminal nucleotidelinked to a cholesteryl derivative or dodecanoic acid bisdecylamidegroup, a 2′-deoxy-2′-fluoro modified nucleotide, a 2′-deoxy-modifiednucleotide, a locked nucleotide, an abasic nucleotide, a2′-amino-modified nucleotide, a 2′-alkyl-modified nucleotide, amorpholino nucleotide, a phosphoramidate, and a non-natural basecomprising nucleotide.
 5. The dsRNA of claim 3, comprising at least one2′-O-methyl modified nucleotide and at least one2′-deoxythymidine-3′-phosphate nucleotide comprising a5′-phosphorothioate group.
 6. The dsRNA of claim 1, further comprising aligand.
 7. The dsRNA of claim 1, further comprising a ligand conjugatedto the 3′-end of the sense strand of the dsRNA.
 8. A composition forinhibiting expression of a GNAQ gene comprising the dsRNA of claim 1 andpharmaceutical formulation.
 9. The composition of claim 8, wherein thepharmaceutical formulation is a lipid formulation.
 10. The compositionof claim 8, wherein the pharmaceutical formulation is a(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl4-(dimethylamino)butanoate (MC3) comprising formulation.
 11. An isolatedcell containing the dsRNA of claim
 1. 12. A vector comprising anucleotide sequence that encodes at least one strand of the dsRNA ofclaim
 1. 13. The dsRNA of claim 1, wherein said dsRNA, upon contact witha cell expressing said GNAQ, inhibits expression of said GNAQ gene by atleast 40% compared to a cell not so contacted.
 14. The dsRNA of claim 1,wherein the dsRNA has an IC50 of less than 10 pM.
 15. A method ofinhibiting GNAQ expression in a cell, the method comprising: (a)introducing into the cell the dsRNA of claim 1; and (b) maintaining thecell produced in step (a) for a time sufficient to obtain degradation ofthe mRNA transcript of a GNAQ gene, thereby inhibiting expression of theGNAQ gene in the cell.
 16. The method of claim 15, wherein expression isinhibited by at least 20%, 40%, 60%, or at least 80%.
 17. A method oftreating a disorder mediated by GNAQ expression comprising administeringto a human in need of such treatment a therapeutically effective amountof the dsRNA of claim
 1. 18. The method of claim 17, wherein the humanhas uveal melanoma, cutaneous melanoma, Blue nevi, Nevi of Ota, a smalllung tumor, or a neuroendocrine tumor.
 19. The method of claim 17,further comprising administering an additional composition.
 20. Themethod of claim 17, further comprising administering a second dsRNA. 21.The dsRNA of claim 1, wherein each strand comprises a 3′ overhangconsisting of dTdT.
 22. The dsRNA of claim 1, wherein the sense strandcomprises SEQ ID NO: 1395 and the antisense strand comprises SEQ ID NO:1396.
 23. The dsRNA of claim 1, wherein the sense strand sequenceconsists of SEQ ID NO: 1395, and the antisense strand comprises SEQ IDNO:
 1396. 24. The dsRNA of claim 1, wherein the sense strand sequenceconsists of SEQ ID NO: 1553 (GucAGGAcAcAucGuucGAdTdT) and the antisensestrand consists of SEQ ID NO: 1554 (UCGAACGAUGUGUCCUGACdTdT) whereineach strand includes 2′-O-methyl ribonucleotides as indicated by a lowercase letter “u” and “c”.